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Sample records for cell dna synthesis

  1. DNA-repair synthesis in ataxia telangiectasia lymphoblastoid cells

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    Ford, M.D.; Houldsworth, J.; Lavin, M.F. (Queensland Univ., Brisbane (Australia). Dept. of Biochemistry)

    1981-12-01

    The ability of a number of Epstein-Barr virus-transformed lymphoblastoid cells from ataxia telangiectasia (AT) patients to repair ..gamma..-radiation damage to DNA was determined. All of these AT cells were previously shown to be hypersensitive to ..gamma..-radiation. Two methods were used to determine DNA-repair synthesis: isopycnic gradient analysis and a method employing hydroxyurea to inhibit semiconservative DNA synthesis. Control, AT heterozygote and AT homozygote cells were demonstrated to have similar capacities for repair of radiation damage to DNA. In addition at high radiation doses (10-40 krad) the extent of inhibition of DNA synthesis was similar in the different cell types.

  2. Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells

    International Nuclear Information System (INIS)

    Seki, Shuji; Hosogi, Nobuo; Oda, Takuzo

    1984-01-01

    In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97 % by aphidicolin at 10 μg/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30 % and 90 % depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90 % at 100 μg/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 μg/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase α and a non-α DNA polymerase (possibly DNA polymerase β), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase α in UDS favored DNA synthesis in the intranucleosomal region. (author)

  3. Effects of inhibitors of DNA synthesis and protein synthesis on the rate of DNA synthesis after exposure of mammalian cells to ultraviolet light

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Dahle, D.B.; Meechan, P.J.; Carpenter, J.G.

    1981-01-01

    Chinese hamster V-79 cells were treated with metabolic inhibitors of DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J m -2 . After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [ 14 C]thymidine into acid-insoluble material. A 2.5 or 5.0h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of the cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involvement of an inducible DNA repair process. (Auth.)

  4. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

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

    1990-01-01

    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

  5. ATP-independent DNA synthesis in Vaccinia-infected L cells

    International Nuclear Information System (INIS)

    Berger, N.A.; Kauff, R.A.; Sikorski, G.W.

    1978-01-01

    Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl 2 . DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes. (Auth.)

  6. γ-irradiation induces radioresistant DNA synthesis in HeLa cells

    International Nuclear Information System (INIS)

    Synzynys, B.I.; Aminev, A.G.; Konstantinova, S.A.; Saenko, A.S.

    1987-01-01

    Cells of suspension HeLa culture at the logarithmic phase of growth were exposed to 60 Co-γ-rays (5 Gy), incubated in the nutritious medium, and in 4 h subjected to repeated irradiation: the dose-response function and the dynamics of DNA synthesis inhibition were determined. It was shown that DNA synthesis was inhibited to a lesser extent after preirradiation, in other words, DNA synthesis was radioresistant. A correlation between this synthesis and reproductive cell death is discussed

  7. Radiation-induced depression of DNA synthesis in cultured mammalian cells

    International Nuclear Information System (INIS)

    Povirk, L.F.

    1977-01-01

    A 313-nm light source was constructed in order to study the mechanisms by which ultraviolet and ionizing radiations inhibit DNA synthesis. It was found that in CHO, MDBK and HeLa cells, grown for one generation in the DNA sensitizer bromodeoxyuridine (BrdUrd), 313-nm light inhibited DNA synthesis with a pattern similar to that of the effect of x-rays on normal cells. A biphasic dose response curve for inhibition of total synthesis was observed, with a sensitive component representing depression of initiation of new replicons and a resistant component representing interference with elongation of replicons already growing at the time of irradiation. Since the BrdUrd plus 313-nm light treatment produces DNA lesions similar to those produced by x-rays (base damage, strand breaks, crosslinks) these results suggest that the effect of x-rays on DNA synthesis is mediated by DNA damage. In experiments with synchronized cells, it was found that in cells in which about half the chromosomes had incorporated BrdUrd, 313-nm light inhibited replication of the BrdUrd-containing DNA, but had no effect on the replication of the unsubstituted DNA in the same cell. Thus the information that DNA is damaged appears to be propagated along the DNA molecule from the sites of damage to the replication initiation sites as some kind of conformational change, possibly a relaxation of superhelical tension. Target theory calculations suggest that a single DNA lesion prevents the initiation of several adjacent replicons

  8. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Ciarrocchi, G.; Linn, S.

    1978-01-01

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  9. Effects of DNA polymerase inhibitors on replicative and repair DNA synthesis in ultraviolet-irradiated HeLa cells

    International Nuclear Information System (INIS)

    Morita, T.; Nakamura, H.; Tsutsui, Y.; Nishiyama, Y.; Yoshida, S.

    1982-01-01

    Aphidicolin specifically inhibits eukaryotic DNA polymerase α, while 2',3'-dideoxythymidine 5'-triphosphate (d 2 TTP) inhibits DNA polymerase ν and ν but not α. 1-ν-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) inhibits both DNA polymerase α and ν although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-ν-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2',3'-dideoxythimidine (d 2 Thd, a precursor of d 2 TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d 2 Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d 2 TTP, was microinjected directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d 2 TTP effectively inhibited repair synthesis. The microinjection of d 2 TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis

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

    International Nuclear Information System (INIS)

    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 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 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

  11. Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Barenfeld, L.S.; Pleskach, N.M.; Bildin, V.N.; Prokofjeva, V.V.; Mikhelson, V.M.

    1986-01-01

    The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [ 3 H]DNA in alkaline sucrose gradients or by direct assay of the amount of [ 3 H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)

  12. Dissociation of histone and DNA synthesis in x-irradiated HeLa cells

    International Nuclear Information System (INIS)

    Bases, R.; Mendez, F.

    1971-01-01

    Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1,000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal

  13. DNA synthesis in permeabilized WI38 and MRC5 cells

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Carpenter, J.G.

    1980-01-01

    DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident

  14. DNA synthesis and uv resistance in Escherichia coli K12 cells

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    Slezarikova, V [Slovenska Akademia Vied, Bratislava (Czechoslovakia). Vyskumny Ustav Onkologicky

    1976-01-01

    The influence was studied of preirradiation inhibition of proteosynthesis by amino acids starvation on survival and DNA synthesis in E. coli K 12 cells, which differ by their genetic features with regard to a certain type of repair. The surviving fraction was studied by appropriate dilution of cell suspension and spreading on agar plates. DNA synthesis was investigated by the incorporation of thymine-2-/sup 14/C. In our conditions a correlation was found between cell survival and the resistance of DNA replication to UV radiation in cells proficient in excision and post-replication repair. This correlation was not found in the excision deficient strain. It is concluded that enhanced resistance of DNA replication is not a sufficient condition for enhanced cell resistance.

  15. In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

    International Nuclear Information System (INIS)

    Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M.

    1988-01-01

    Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K m values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 μM. For UV-induced DNA repair synthesis, the apparent K m values were substantially lower, ranging from 0.11 to 0.44 μM for AG1518 cells and from 0.06 to 0.24 μM for IMR-90 cells. Recent data implicate DNA polymerase δ in UV-induced repair synthesis and suggest that DNA polymerases α and δ are both involved in semiconservative replication. They measured K m values for dGTP and dTTP for polymerases α and δ, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K m values for DNA polymerase δ are much greater than the K m values for UV-induced repair synthesis, suggesting that when polymerase δ functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K m values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K m for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo

  16. Herpes virus and viral DNA synthesis in ultraviolet light-irradiated cells

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    Coppey, J; Nocentini, S [Institut du Radium, 75 - Paris (France). Lab. Curie

    1976-07-01

    The rate of virus DNA synthesis and the production of infectious virus are impaired in stationary monkey kidney CV-I cells irradiated with u.v. before infection with herpes simplex virus (HSV). The inhibition of HSV multiplication is due to u.v.-induced damage in cell DNA. CV-I cells recover their capacity to support HSV growth during the 40 to 48 h after irradiation, and the final virus yield is enhanced by factor of 10. The time course of the recovery is similar to that of the excision repair process occurring in u.v.-irradiated mammalian cells. Caffeine, hydroxyurea and cycloheximide inhibit the recovery. Fluorodeoxyuridine is without effect. A small but significant amount of labelled dThd coming from irradiated cell DNA is incorporated into virus DNA. HSV specified thymidine kinase seems to be more effective for virus DNA synthesis in irradiated than in control cells.

  17. DNA-synthesis inhibition and repair DNA-synthesis in CHO Ade- C cells: An alternative approach to genotoxicity testing

    International Nuclear Information System (INIS)

    Slamenova, D.; Papsova, E.; Gabelova, A.; Dusinska, M.; Collins, A.; Wsolova, L.

    1997-01-01

    We describe an alternative assay to determine genotoxicity. Its main feature is that it combines two measures in a single experiment; the inhibition of replicative DNA synthesis together with the stimulation of DNA repair. We show that, in tests of four different genotoxic agents, the assay gives results that are entirely consistent with what is known about the mode of action of these agents. In addition, we have demonstrated that chemical carcinogens requiring metabolic activation can be examined using a standard procedure of incubation with a microsomal activating fraction. We consider the combined assay for DNA synthesis inhibition and repair synthesis to be a useful way for the rapid pre-screening of chemicals suspected of genotoxic activity on the level of mammalian cells. (author)

  18. Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

    International Nuclear Information System (INIS)

    Lee, E.W.; Johnson, J.T.; Garner, C.D.

    1989-01-01

    Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [ 3 H]thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 μM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [ 3 H]thymidine triphosphate into DNA up to 24 μM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase α, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase 1, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply the p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase α, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause

  19. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Science.gov (United States)

    Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

    2011-01-31

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  20. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    2011-01-01

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  1. The rate of DNA synthesis in normal human and ataxia telangiectasia cells after exposure to X-irradiation

    International Nuclear Information System (INIS)

    Wit, J. de; Bootsma, D.; Jaspers, N.G.J.; Rijksverdedigingsorganisatie TNO, Rijswijk

    1981-01-01

    The rate of DNA synthesis was studied in normal cell strains and in strains from patients suffering from the inherited disorder ataxia telangiectasia (AT). After exposure to relatively low doses of oxic X-rays (0- 4 krad) DNA synthesis was depressed in AT cell strains to a significantly lesser extent than in normal cells. This response was observed in both an excision-deficient and an excision-proficient strain. In contrast, there was no difference in DNA-synthesis inhibition between AT and normal cells after UV exposure. After X-irradiation of cells from patients with xeroderma pigmentosum, both complementation group A and XP variants, the observed rate of DNA synthesis was equal to that in normal cells. An exception was the strain XP3BR which has been shown to be X-ray-sensitive. This strain exhibited diminished DNA synthesis inhibition after X-ray doses below 1 krad. These data suggest a relationship between hypersensitivity to X-rays and diminished depression of DNA synthesis. (orig.)

  2. Postirradiation DNA synthesis is inversely related to cell survival

    International Nuclear Information System (INIS)

    Kapiszewska, M.; Lange, C.S.

    1987-01-01

    Postirradiation (PI) events which might lead to cellular reproductive death or survival were studied in L5178Y-S (LY-S) cells. PI incubation at 25 0 C protects LY-S cells against the PLD fixation which takes place at 37 0 C. An optimal condition for the repair of PLD is 1h at 37 0 C followed by 4h holding at 25 0 C prior to the second half of a split dose, or 5L holding at 25 0 C without a 37 0 C incubation. Longer incubations at 37 0 C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37 0 C was observed only during the first 30 min; thereafter, /sup 3/H-dThd incorporation was higher than in unirradiated controls. This excess synthesis effect was removed by shifting irradiated cells to 25 0 C holding. The inhibition observed at 25 0 C was reversed by shifting to 37 0 C. Thus the degree of postirradiation DNA synthesis is inversely related to PLD/SLD repair. DNA filter elution shows complete SSB repair by 3h at both temperatures (with faster kinetics at 37 0 C), and DSB repair plateaus at 80% (37 0 C) and 60% (25 0 C) after 90 min

  3. Relative ultraviolet radiation sensitivity of certain functions of polyoma virus. Stimulation of cell DNA synthesis

    International Nuclear Information System (INIS)

    Barra, Yves; Imbert, Jean; Planche, Jacqueline; Meyer, Georges.

    1977-01-01

    Peritoneal Mouse macrophages were used to study the stimulation of cell DNA synthesis by polyoma virus. Using ultraviolet-irradiated polyoma virus, it was possible to show a difference between the inactivation of infectivity and of induction of DNA synthesis. By statistical analysis of these two phenomena it was found that 39% of the viral genome is necessary for the induction of cell DNA synthesis [fr

  4. Murine scid cells complement ataxia-telangiectasia cells and show a normal port-irradiation response of DNA synthesis

    International Nuclear Information System (INIS)

    Komatsu, K.; Yoshida, M.; Okumura, Y.

    1993-01-01

    The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a deficit in lymphoid variable-(diversity)-joining (V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Significantly reduced D 0 and n values were demonstrated in scid cells and were similar to ataxia-telangiectasia (AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Results indicate that the radiobiological character of scid is similar to AT but is presumably caused by different mechanisms. (author)

  5. Desoxyribonucleic acid (DNA) synthesis in vitro by thymus and spleen cells of the rat after hyperthermia

    Energy Technology Data Exchange (ETDEWEB)

    Tempel, K.; Spath, A.

    1988-03-01

    The inhibition of the semiconservative and restorative DNA synthesis caused by hyperthermia (30 to 60 min, 43/sup 0/C) was significantly higher in spleen cells than in thymus cells. The DNA repair synthesis of thymus cells measured at 37/sup 0/C was increased by about two times the initial value after a pre-incubation of 30 to 90 min and 30 to 60 min, respectively, with 37 and 43/sup 0/C, respectively. Under the same conditions, the /sup 3/H-thymidine incorporation into the DNA of spleen cells diminished proportionally to the pre-incubation time after a pre-incubation of 30 and 45 min, respectively, with 43 and 37/sup 0/C, respectively. When hyperthermia and inhibitors of DNA synthesis or DNA repair (hydroxyurea, 1-..beta..-D-arabinofuranosylcytosine, 3', 5'-didesoxythymidine, and 3-aminobenzamide) were combined, overadditive effects - without cellspecific particularities - were seen only in the case of 3-aminobenzamide. Only in thymus cells, the inhibitor of DNA topoisomerase II novobiocin caused an overadditive reinforcement of the inhibition induced by hyperthermia of the semiconservative DNA synthesis. The stimulation of DNA repair synthesis in thymus cells caused by novobiocin with the aid of DNA polymerase ..beta.. could be compensated by hyperthermia. The sedimentation of thymus and spleen cell nucleoids was increased after hyperthermia. The results suggest a special importance of DNA topology and of the DNA polymerase ..beta.. activity for the cellular effect of hyperthermia.

  6. DNA synthesis during development and proliferation of glial cells in organotypic rat cerebellar culture

    International Nuclear Information System (INIS)

    Renkawek, K.

    1977-01-01

    DNA synthesis was investigated in glial cells in vitro with 3 H thymidine in concentration 1 μCi/ml medium. Incorporation of isotope into the glial nuclei has been found both in the explant (7-21%) and in the outgrowth (22-56%). DNA synthesis was dependent on the age of culture and due to the contact inhibition in the outgrowth. Results point out that marked DNA synthesis is a characteristic feature of glia differentiation and of reactive character of glial cells in vitro. (author)

  7. Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

    International Nuclear Information System (INIS)

    Vikhanskaya, F.L.; Khrebtukova, I.A.; Manuilova, E.S.

    1985-01-01

    A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m 2 ) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m 2 ) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair. (orig.)

  8. Bronchoalveolar lavage fluid from normal rats stimulates DNA synthesis in rat alveolar type II cells

    International Nuclear Information System (INIS)

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

    1989-01-01

    Proliferation of alveolar type II cells after lung injury is important for the restoration of the alveolar epithelium. Bronchoalveolar lavage fluid (BALF) may represent an important source of growth factors for alveolar type II cells. To test this possibility, BALF fluid was collected from normal rats, concentrated 10-fold by Amicon filtration, and tested for its ability to stimulate DNA synthesis in rat alveolar type II cells in primary culture. BALF induced a dose-dependent increase in type II cell DNA synthesis resulting in a 6-fold increase in [3H]thymidine incorporation. Similar doses also stimulated [3H]thymidine incorporation into rat lung fibroblasts by 6- to 8-fold. Removal of pulmonary surface active material by centrifugation did not significantly reduce the stimulatory activity of BALF for type II cells. The stimulation of type II cell DNA synthesis by BALF was reduced by 100% after heating at 100 degrees C for 10 min, and by approximately 80% after reduction with dithiothreitol, and after trypsin treatment. Dialysis of BALF against 1 N acetic acid resulted in a 27% reduction in stimulatory activity. The effect of BALF in promoting type II cell DNA synthesis was more pronounced when tested in the presence of serum, although serum itself has very little effect on type II cell DNA synthesis. When BALF was tested in combination with other substances that stimulate type II cell DNA synthesis (cholera toxin, insulin, epidermal growth factor, and acidic fibroblast growth factor), additive effects or greater were observed. When BALF was chromatographed over Sephadex G150, the activity eluted with an apparent molecular weight of 100 kDa

  9. DNA repair synthesis in human fibroblasts requires DNA polymerase delta

    International Nuclear Information System (INIS)

    Nishida, C.; Reinhard, P.; Linn, S.

    1988-01-01

    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

  10. Lethality and the depression on DNA synthesis in UV-irradiated normal human and xeroderma pigmentosum cells

    Energy Technology Data Exchange (ETDEWEB)

    Shinohara, K. (Kobe Univ. (Japan). School of Medicine)

    1983-12-01

    Ultraviolet radiation suppresses the semiconservative DNA replication in mammalian cells. The rate of DNA synthesis is initially depressed and later recovers after low doses of UV radiation in human cells. Such a response is more sensitive to UV radiation in cells derived from patients with xeroderma pigmentosum (XP) than that in normal human cells. The relative rate of DNA synthesis is not always correlated with cell survival because, unlike cell survival, the dose-response curve of the relative rate of DNA synthesis shows the biphasic nature of the sensitivity. In the experiments reported herein, the total amount (not the rate) of DNA synthesized during a long interval of incubation which covers the period of inhibition and recovery (but not longer than one generation time) after irradiation with various doses of UV radiation was examined in normal human and XP cells, and was found to be well correlated with cell survival in all the cells tested.

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

    International Nuclear Information System (INIS)

    Glushankova, N.A.

    1986-01-01

    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 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

  12. Spontaneous unscheduled DNA synthesis in human lymphocytes

    International Nuclear Information System (INIS)

    Forell, B.; Myers, L.S. Jr.; Norman, A.

    1979-01-01

    The rate of spontaneous unscheduled DNA synthesis in human lymphocytes was estimated from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. The contribution of scheduled DNA synthesis to the observed incorporation was reduced by inhibiting replication with hydroxyurea and by separating freshly replicated single-stranded DNA (ss-DNA) from repaired ds-DNA by column chromatography. The residual contribution of scheduled DNA synthesis was estimated by observing effects on thymidine incorporation of: (a) increasing the rate of production of apurinic sites, and alternatively, (b) increasing the number of cells in S-phase. Corrections based on estimates of endogenous pool size were also made. The rate of spontaneous unscheduled DNA synthesis is estimated to be 490 +- 120 thymidine molecules incorporated per cell per hour. These results compare favorably with estimates made from rates of depurination and depyrimidination of DNA, measured in molecular systems if we assume thymidine is incorporated by a short patch mechanism which incorporates an average of four bases per lesion

  13. Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA

    International Nuclear Information System (INIS)

    Setlow, B.; Hand, A.R.; Setlow, P.

    1991-01-01

    Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores

  14. Decreased UV-induced DNA repair synthesis in peripheral leukocytes from patients with the nevoid basal cell carcinoma syndrome

    International Nuclear Information System (INIS)

    Ringborg, U.; Lambert, B.; Landergen, J.; Lewensohn, R.

    1981-01-01

    The uv-induced DNA repair synthesis in peripheral leukocytes from 7 patients with the nevoid basal cell carcinoma syndrome was compared to that in peripheral leukocytes from 5 patients with basal cell carcinomas and 39 healthy subjects. A dose response curve was established for each individual, and maximum DNA repair synthesis was used as a measure of the capacity for DNA repair. The patients with the nevoid basal cell carcinoma syndrome had about 25% lower level of maximum DNA repair synthesis as compared to the patients with basal cell carcinomas and control individuals. The possibility that DNA repair mechanisms may be involved in the etiology to the nevoid basal cell carcinoma syndrome is discussed

  15. DNA synthesis in vitro in human fibroblast preparations

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, W.K.

    1983-01-01

    When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15-30 min incubation at 37/sup 0/C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37/sup 0/C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 hr at 37/sup 0/C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

  16. Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Ventura, A M; Ortega, J M; Schumacher, R I; Meneghini, R

    1987-01-01

    In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. Using metabolic inhibitors, it has been shown that (1) even the low repair rate exhibited by V79 cells is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of small replicons.

  17. Effect of Vaccinia virus infection on poly(ADP-ribose)synthesis and DNA metabolism in different cells

    Energy Technology Data Exchange (ETDEWEB)

    Topaloglou, A.; Ott, E.; Altmann, H. (Oesterreichisches Forschungszentrum Seibersdorf G.m.b.H. Inst. fuer Biologie); Zashukhina, G.D.; Sinelschikova, T.A. (AN SSSR, Moscow. Inst. Obshchej Genetiki)

    1983-07-14

    In Chang liver cells and rat spleen cells infected with Vaccinia virus, DNA synthesis, repair replication after UV irradiation and poly(ADP-ribose)(PAR) synthesis were determined. In the time post infection semiconservative DNA synthesis showed only a slight reduction. DNA repair replication was not very different from controls 4 hours p.i. but was enhanced 24 hours after infection compared to noninfected cells. PAR synthesis was also not changed very much 4 hours p.i. but was decreased significantly after 24 hours. The determination of radioactivity resulting from /sup 3/H-NAD, showed a marked reduction of PAR in the spacer region of chromatin 24 hours p.i., but in addition, PAR located in the core region, was reduced, too.

  18. Effects of gamma- and UV-radiation on DNA synthesis in permeable cells of Bacillus stearothermophilus

    International Nuclear Information System (INIS)

    Trofimenko, A.F.; Vorob'eva, A.M.; Gaziev, A.I.

    1981-01-01

    It was shown that the most of the DNA synthesis is repaired in permeable cells of Bacillus stearothermophilus not affected by injurious agents. γ-irradiation stimulates the reparative synthesis and degradation of DNA whereas UV-radiation decreases the activity of these processes. The reason for such an unusual response of thermophiles to irradiation lies perhaps in high temperatures at which the cells exist

  19. Simple Laboratory methods to measure cell proliferation using DNA synthesis property

    Directory of Open Access Journals (Sweden)

    Madhavan H N

    2007-01-01

    Full Text Available This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid. DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

  20. DNA synthesis and cell survival after X-irradiation of mammalian cells treated with caffeine or adenine

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Carpenter, J.G.; Dahle, D.B.

    1978-01-01

    The expression of the transient depression in the rate of DNA synthesis normally observed after exposure of randomly-dividing Chinese hamster V-79 or Chinese hamster CHO cells to ionizing radiation could be postponed by a post-irradiation treatment with 1.0 to 2.0 mM adenine or 1.5 mM caffeine. Caffeine may exert its effect by creating additional sites for replication in irradiated cells. Cells treated with caffeine or adenine for 2 or 4 hours after exposure to 3000 rad of 300 kVp X-rays exhibited depressed synthesis only after the removal of caffeine or adenine. These alterations in the timing of the X-ray-induced depression of the rate of DNA synthesis had no effect on X-ray-induced cell killing. Although a 4 hour post-irradiation treatment of randomly-dividing Chinese hamster V-79 cells with 1.0 or 2.0 mM caffeine potentiated X-ray-induced cell killing, this reduction in survival was due primarily to effects on cells not in S-phase. (author)

  1. Recovery from DNA synthesis in V 79 chinese hamster cells irradiated with UV light

    International Nuclear Information System (INIS)

    Ventura, A.M.

    1987-01-01

    Mammalian cells recover from DNA synthesis inhibition by UV light before most of the pyrimidine dimers have been removed from the genome. Most of the rodent cells show a deficient dimer excision repair compared with normal human fibroblasts. Despite this fact they recover efficiently from DNA synthesis inhibition after UV. In Chinese hamster V 79 cells was found that this recovery takes place in the absence of a significant excision repair, and it seems to be directly coupled to a recovery in the rate of movement of the replication fork. 120 refs, 31 figs. (author)

  2. The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells ''in vitro''

    International Nuclear Information System (INIS)

    Klein, W.; Altmann, H.; Kocsis, F.; Egg, D.; Guenther, R.

    1978-03-01

    ''In vitro'' experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A 1 , B 1 , E 1 , E 2 and Fsub(2α) were tested in concentrations of 10 pg, 5 ng and 2,5μg per ml cell suspension. DNA synthesis was significantly increased by PgFsub(2α) in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgE 1 and PgE 2 at 5 ng/ml and at 2,5 μg/ml but increased by PgFsub(2α) in the two lower concentrations. The rejoining of DNA-strand breaks after gamma-irradiation was slightly reduced by PgE 1 , PgE 2 and PgF 2 at 2,5 μg/ml. (author)

  3. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    International Nuclear Information System (INIS)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

    1991-01-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

  4. Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

    International Nuclear Information System (INIS)

    Sugaya, Shigeru; Nakanishi, Hiroshi; Tanzawa, Hideki; Sugita, Katsuo; Kita, Kazuko; Suzuki, Nobuo

    2005-01-01

    Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested

  5. Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

    Energy Technology Data Exchange (ETDEWEB)

    Sugaya, Shigeru [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Nakanishi, Hiroshi [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Tanzawa, Hideki [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Sugita, Katsuo [Department of Clinical Medicine, Faculty of Education, Chiba University, 1-33 Yayoi, Inage-ku, Chiba 263-8522 (Japan); Kita, Kazuko [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Suzuki, Nobuo [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan)]. E-mail: nobuo@faculty.chiba-u.jp

    2005-10-15

    Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested.

  6. The effect of caffeine and adenine on radiation induced suppression of DNA synthesis, and cell survival

    International Nuclear Information System (INIS)

    Wilcoxson, L.T.; Griffiths, T.D.

    1984-01-01

    Exposure of cultured mammalian cells to ionizing radiation or UV light results in a transient decrease in the rate of DNA synthesis. This depression in synthetic rate may be attenuated or deferred via a post-irradiation treatment with caffeine or adenine. It has been suggested that this attenuation may increase the fixation of damage and, therefore, increase radiation sensitivity. However, it has been previously reported that, for V79 cells treated with caffeine or adenine, no correlation exists between the extent of depression and cell survival. The present investigation expands upon these findings by examining the effect of caffeine or adenine post-irradiation treatment on two cell lines with normal UV sensitivity, mouse 3T3 and CHO AA8 cells, and one UV sensitive cell line, CHO UV5 cells. Both caffeine and adenine have been found to reduce, or delay, the suppression in DNA synthesis in all three cell lines. Surprisingly, caffeine appeared to induced even the UV5 cells to recover DNA synthetic ability. The amount of reduction in suppression of DNA synthesis, however, varies between the different cell lines and no consistent relationship with cell survival has emerged

  7. Escape from X-ray-induced arrest for lens cells stimulated from quiescence: time relationship to RNA, protein, and DNA synthesis

    International Nuclear Information System (INIS)

    Lindgren, A.L.; Miller, R.C.; Guernsey, D.L.; Riley, E.F.

    1988-01-01

    Quiescent cells of the central zone region of the rat lens epithelium were stimulated to enter the proliferation cycle by wounding. RNA synthesis and a corresponding increase in poly(A)+/total RNA reached a peak by Hour 4. Cells progressed into the G1B compartment by Hour 10. A rise in protein synthesis began at Hour 8, and onset of DNA synthesis occurred by Hour 14. The timing of cell cycle progression that allowed escape from a dose of X irradiation that completely inhibited DNA synthesis was investigated. A growth-arrest point was identified at Hour 9 where 10 GY of X irradiation given before, but not after, completely inhibited earliest responding cells from entering DNA synthesis on schedule. Increased quantities of cells entered DNA synthesis on schedule as timing of the X irradiation was moved closer to the end of G1. Based on time relationships, the rise in protein synthesis is correlated with the sufficient event for the escape

  8. Unscheduled DNA synthesis and elimination of DNA damage in liver cells of. gamma. -irradiated senescent mice

    Energy Technology Data Exchange (ETDEWEB)

    Gaziev, A.I.; Malakhova, L.V. (AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki)

    1982-10-01

    The level of 'spontaneous' and ..gamma..-radiation-induced DNA synthesis which is not inhibited with hydroxyurea (unscheduled synthesis) is considerably lower in hepatocytes of 18-22-month-old mice than that of 1.5-2-month-old mice. The dose-dependent increase (10-300 Gy) of unscheduled DNA synthesis (UDS) in hepatocytes of senescent mice is higher than in young animals. The elimination of damage in DNA of ..gamma..-irradiated hepatocytes (100 Gy) was examined by using an enzyme system (M. luteus extract and DNA-polymerase I of E. coli). It was found that the rate of elimination of the DNA damage in hepatocytes of 20-month-old mice is lower than that of 2-month-old mice although the activities of DNA-polymerase ..beta.. and apurinic endonuclease remain equal in the liver of both senescent and young mice. However, the nucleoids from ..gamma..-irradiated liver nuclei of 2-month-old mice are relaxed to a greater extent (as judged by the criterion of ethidium-binding capacity) than those of 20-month-old mice. The results suggest that there are limitations in the functioning of repair enzymes and in their access to damaged DNA sites in the chromatin of senescent mouse liver cells.

  9. Action of caffeine on x-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Tolmach, L.J.; Jones, R.W.; Busse, P.M.

    1977-01-01

    Treatment of HeLa S3 cells with 1 mM caffeine delays progression through G1 by 1.5 hours but causes no other detectable inhibition of cell progression; it sometimes results in a large stimulation of thymidine incorporation. When this concentration is applied to cells that have been irradiated with 1-krad doses of 220-kV x rays, there is a marked suppression of both the inhibition of DNA synthesis and G2 arrest induced by the radiation. Larger doses require higher concentrations of caffeine to suppress the inhibition of DNA synthesis. Delaying addition until the rate of synthesis is at its minimum (1.5 hours after irradiation with 1 krad) results in a slightly accelerated recovery of the rate. Treatment before or during irradiation is without effect on the inhibition. Removal of the caffeine as late as 6 hours after its addition at the time of irradiation results in a prompt inhibition in DNA synthesis that mimics that observed immediately after irradiation in the absence of caffeine. These findings raise the possibility that the depression in rate of DNA systhesis might not result from radiation damage introduced into the replicon initiation system, but rather may be an indirect consequence of damage residing elsewhere in the irradiated cell

  10. DNA synthesis in HeLa cells and isolated nuclei after treatment with an inhibitor of spermidine synthesis, methyl glyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Krokan, H; Eriksen, A

    1977-02-01

    Addition of methyl glyoxal bis(guanylhydrazone) to HeLa S3 suspension cultures resulted in increased putrescine levels and decreased spermidine and spermine levels preceding a drop in incorporation of [3H]thymidine, [3H]uridine and [14C]leucine into macromolecules. When putrescine, spermidine, spermine or cadaverine was added simultaneously with methyl glyoxal bis(guanylhydrazone), the drug had no detectable effect on the synthesis of macromolecules. In nuclei isolated from cells treated with methyl glyoxal bis(guanylhydrazone) the reduction in the rate of DNA synthesis was equal to the reduction of [3H]thymidine incorporation in the corresponding whole cells. The capability of the nuclei to synthesize DNA could not be restored by adding spermidine or spermine to the system in vitro. The rate of DNA chain elongation was only reduced slightly by methyl glyoxal bis(guanylhydrazone) indicating that decreased levels of spermidine and spermine lead to a decrease in the number of replication units active in DNA synthesis within each cell.

  11. Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light

    International Nuclear Information System (INIS)

    Painter, R.B.

    1985-01-01

    The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion. (orig.)

  12. Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

    International Nuclear Information System (INIS)

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Unscheduled DNA synthesis has been measured in human fibroblasts under conditions of reduced rates of conversion of NAD to poly)ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of uv induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with uv or N-methyl-N'-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis

  13. Assessment of DNA synthesis in Islet-1+ cells in the adult murine heart

    International Nuclear Information System (INIS)

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

    2015-01-01

    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 + ) 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 + 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 ( 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 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 + cells. Whereas Islet − 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 + 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

  14. Autoradiographic study of gamma-ray induced unscheduled DNA synthesis in bean root meristem cells

    International Nuclear Information System (INIS)

    Liu Zhenshen; Qiu Quanfa; Chen Dongli

    1989-01-01

    The gamma-ray induced unscheduled DNA synthesis in root meristem cells of Vica faba was studied autoradiographically by calculating the number of cells with different 3H-thymidine labelling degree. It was found that the level of unscheduled synthesis in cells with intermediate dose (500 R) irradiation was higher than that in cells with lower dose (250 R) irradiation; however, higher dose (1000 R) irradiation would inhibit the reparative replication

  15. Sensitization of human cells by inhibitors of DNA synthesis following the action of DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Filatov, M.V.; Noskin, L.A. (Leningrad Inst. of Nuclear Physics, Gatchina (USSR))

    1983-08-01

    Inhibitors of DNA synthesis 1-..beta..-arabinofuranosylcytosine (Ac) and hydroxyurea (Hu) taken together drastically sensitized human cells to the killing effect of DNA-damaging agents. For UV-irradiation this sensitization depended on the cells' ability for excision repair. By using viscoelastometric methods of measurement of double-strand breaks (DSB) in the genome, it was established that the first DSB were generated after incubation of the damaged cells in the mixture of inhibitors at about the same dose when sensitization appeared. A scheme is proposed to describe molecular events associated with the phenomenon studied. 35 refs.

  16. Radiation metagenesis and inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Dubinina, L.G.; Sergievskaya, S.P.; Kurashova, Z.I.; Dubinin, N.P.

    1983-01-01

    The study of modification of radiation mutagenesis and inhibition of the DNA synthesis by means of 1-β-D arabinofuranosylcytosine (ara-C) is carried out. It is shown that ara-C-acting on chromosomes in the G 1 phase and G 2 phase does not cause mutations in the C capillaris cells. The modification by means of ara-C radiation effect in the G 1 phase and G 2 phase correlates with duration and time of administering ara-C before and after irradiation. A new form of ara-C DNA synthesis inhibitor interaction with mutation processes has been found out. Protective effect of the DNA synthesis inhibitor (ara-C) from mutageneous radiation effect is stressed. Sensibilization of the radiation mutagenesis during cell treafment by the DNA synthesis inhibitor (ara-C) is shown. It is pointed out that emergence of sensibilization or protective effect, i. e. antimutagenesis phenomenon depends on conditions under which the synthesis inhibitor acted in G 1 and G 2 phases

  17. Different effects of inorganic and dimethylated arsenic compounds on cell morphology, cytoskeletal organization, and DNA synthesis in cultured Chinese hamster V79 cells

    Energy Technology Data Exchange (ETDEWEB)

    Ochi, Takafumi; Nakajima, Fumie [Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa (Japan); Fukumori, Nobutaka [Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Hyakuninchou, Shinjyuku (Japan)

    1998-09-01

    Changes in cytoskeletal organization of cultured V79 cells exposed to arsenite and dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, and related changes, such as mitotic arrest and induction of multinucleated cells, were investigated in comparison with their effects on DNA synthesis. DMAA caused mitotic arrest and induction of multinucleated cells with a delay of 12 h relative to the mitotic arrest. By contrast, arsenite at equitoxic concentrations to DMAA was less effective than DMAA in causing mitotic arrest and in inducing multinucleated cells. Post-mitotic incubation of cells arrested in metaphase by 6 h incubation with 10 mM DMAA showed that the incidence of multinucleated cells increased conversely with a rapid decrease in metaphase cells. This suggests that metaphase-arrested cells can escape from metaphase, resulting in the appearance of multinucleated cells. The mitotic arrest caused by DMAA was accompanied by disruption of the microtubule network. By contrast, both arsenite and DMAA did not cause disorganization of actin stress fibers even when incubated at concentrations that caused a marked retardation of cell growth. Cells exposed to arsenite for 6 h showed marked inhibition of DNA synthesis, whereas inhibition by DMAA was not observed. When incubation was prolonged by 18 h, the arsenite-induced inhibition of DNA synthesis was mitigated. By contrast, inhibition of DNA synthesis by DMAA occurred in parallel with an increase in the population of mitotic cells. These results suggest that DMAA caused growth retardation and morphological changes via disruption of the microtubule network, and that arsenite-induced retardation of cell growth and inhibition of DNA synthesis were not attributable to the cytoskeletal changes. (orig.) (orig.) With 7 figs., 31 refs.

  18. Recovery of subchromosomal DNA synthesis in synchronous V-79 Chinese hamster cells after ultraviolet light exposure

    International Nuclear Information System (INIS)

    Meechan, P.J.; Carpenter, J.G.

    1986-01-01

    Previous work obtained from Chinese hamster V-79 cells indicated that, immediately following exposure, UV-induced lesions acted as blocks to elongation of nascent strands, but gradually lost that ability over a 10 h period after exposure to 10 J/m 2 . The work reported herein attempted to examine possible cell cycle mediated alterations in the recovery of DNA synthesis. Kinetic incorporation of radiolabeled thymidine studies indicated that there may have been a more rapid recover of DNA synthesis in cells irradiated in G 1 or G 2 vs cells irradiated in S phase. DNA fiber autoradiograms prepared from synchronous cells indicated that after irradiation in any phase of the cell cycle, the length of newly synthesized DNA was equal to control lengths 1 h after exposure to 5.0Jm 2 (or 1 h after entering S phase for cells irradiated in G 1 or G 2 ). This observed recovery was not solely due to an excision process. No cell cycle mediated difference in the number of dimers induced or removed as a function of cell cycle position was observed. These results appear to be consistent with a continuum of effects, with initiation effects dominating the response at low fluences, gapped synthesis at intermediate fluences and elongation inhibition at high fluences. The fluences at which each event dominates may be cell-line specific. (author)

  19. The acute effects of ionizing radiation on DNA synthesis and the development of antibody-producing cells

    International Nuclear Information System (INIS)

    Harris, G.; Olsen, I.; Cramp, W.A.

    1981-01-01

    Ionizing radiation inhibited the development of specific haemolysin-producing cells (PFC) and depressed the incorporation of ( 3 H) thymidine by rabbit spleen explants responding to SRC in the culture medium. In contrast to these effects, the rates of incorporation of precursors for protein and RNA synthesis were much less affected. The depression of ( 3 H) thymidine incorporation was found to result from a quantitative reduction of new DNA synthesis, without any change in the proportion of labelled cells, at any time after irradiation. The DNA synthesis occurring in these cells preparing to develop antibody-producing capacity was thus radio-sensitive, but the exact nature of the defect resulting from exposure to radiation requires further study. (orig.)

  20. Labelling of Cells Engaged in DNA Synthesis: Autoradiography and BrdU Staining

    DEFF Research Database (Denmark)

    Madsen, Peder Søndergaard

    2010-01-01

    The cell cycle is divided in four phases: G1 phase, S phase (DNA-synthesis), G2 phase (together termed interphase) and M phase (mitosis). Cells that have ceased proliferation enter a state of quiescence called G0. M phase is itself composed of two tightly coupled processes: mitosis, in which...

  1. Cell growth state determines susceptibility of repair DNA synthesis to inhibition by hydroxyurea and 1-beta-D-arabinofuranosylcytosine

    International Nuclear Information System (INIS)

    Mullinger, A.M.; Collins, A.R.; Johnson, R.T.

    1983-01-01

    The effects of inhibitors of replicative DNA synthesis on repair DNA synthesis have been examined by autoradiography in several different cell types and in cells in different growth states. Hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), administered together, influence unscheduled DNA synthesis (UDS) in a manner which is independent of the status of the cell culture (normal or transformed) and of the species, but which is strongly affected by whether the cells are proliferating or quiescent. In proliferating human, Chinese hamster and Microtus cell cultures, UDS is not inhibited by HU and ara C, and may even appear to be stimulated. In quiescent cultures of these cells UDS is reduced by HU and ara C. In cells reseeded from a confluent culture and followed during proliferation and back to quiescence the effect of inhibitors parallels the growth pattern. The results are interpreted in terms of changes in the sizes of endogenous DNA precursor pools; they underline the potential problems associated with quantitating UDS in the presence of inhibitors

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

    Energy Technology Data Exchange (ETDEWEB)

    Weinberger, Florian, E-mail: f.weinberger@uke.de; Mehrkens, Dennis, E-mail: dennis.mehrkens@uk-koeln.de; Starbatty, Jutta, E-mail: starbatty@uke.uni-hamburg.de; Nicol, Philipp, E-mail: Philipp.Nicol@gmx.de; Eschenhagen, Thomas, E-mail: t.eschenhagen@uke.de

    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.

  3. Inhibition of DNA synthesis and radiosensitization effects of thalidomide on esophageal carcinoma TE1 cells

    International Nuclear Information System (INIS)

    Yu Jingping; Sun Suping; Sun Zhiqiang; Sun Meiling; Liu Fenju

    2010-01-01

    Objective: To explore the radiosensitization effect of thalidomide combined with X-ray on esophageal carcinoma TE1 cells. Methods: Cell scratch assay was used to detect the inhibition ability of different concentration of Thalidomide on cell invasion and metastasis. H 3 -TdR incorporation assay was used to investigate the inhibition of DNA synthesis in TE1 cells by treated with Thalidomide singly or combination with X-rays. The colony formation assay was used to analyze the radiosensitization of Thalidomide effect on TE1 cells. Results: Thalidomide had obvious inhibition effect on TE1 cell metastasis, DNA synthesis and colony formation, which were correlated with drug concentration. The values D 0 , D q and SF 2 in TE1 cells were gradually decreased with thalidomide concentration increased. When the concentration of thalidomide was 100μg/ml, the SER D 0 and SER D 0 and SER D q were (1.4±0.2) and (1.5±0.1), respectively, While the concentration of thalidomide was 150 μg/ml, the SER D 0 and SER D q were (1.5±0.2) and (1.8±0.2), respectively. Conclusions: Thalidomide could inhibit TE1 cell invasion, metastasis, DNA synthesis, and significantly enhance the radiosensitizing effect on esophageal carcinoma TE1 cells. (authors)

  4. Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

    International Nuclear Information System (INIS)

    Dreslor, S.L.; Frattini, M.G.

    1987-01-01

    Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N 2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

  5. Timing of initiation of macronuclear DNA synthesis is set during the preceding cell cycle in Paramecium tetraurelia: analysis of the effects of abrupt changes in nutrient level

    International Nuclear Information System (INIS)

    Ching, A.S.L.; Berger, J.D.

    1986-01-01

    In many eukaryotic organisms, initiation of DNA synthesis is associated with a major control point within the cell cycle and reflects the commitment of the cell to the DNA replication-division portion of the cell cycle. In paramecium, the timing of DNA synthesis initiation is established prior to fission during the preceding cell cycle. DNA synthesis normally starts at 0.25 in the cell cycle. When dividing cells are subjected to abrupt nutrient shift-up by transfer from a chemostat culture to medium with excess food, or shift-down from a well-fed culture to exhausted medium, DNA synthesis initiation in the post-shift cell cycle occurs at 0.25 of the parental cell cycle and not at either 0.25 in the post-shift cell cycle or at 0.25 in the equilibrium cell cycle produced under the post-shift conditions. The long delay prior to initiation of DNA synthesis following nutritional shift-up is not a consequence of continued slow growth because the rate of protein synthesis increases rapidly to the normal level after shift-up. Analysis of the relation between increase in cell mass and initiation of DNA synthesis following nutritional shifts indicates that increase in cell mass, per se, is neither a necessary nor a sufficient condition for initiation of DNA synthesis, in spite of the strong association between accumulation of cell mass and initiation of DNA synthesis in cells growing under steady-state conditions

  6. DNA synthesis and degradation in UV-irradiated toluene treated cells of E. coli K12: the role of polynucleotide ligase

    International Nuclear Information System (INIS)

    Strike, P.

    1977-01-01

    Toluene treated cells have been used to study the processes of DNA synthesis and DNA degradation in ultra-violet irradiated Escherichia coli K12. Synthesis and degradation are both shown to occur extensively if polynucleotide ligase is inhibited, and to occur to a much lesser extent if ligase activity is optimal. Extensive UV-induced DNA synthesis in toluene-treated cells requires ATP for the initial incision step, and DNA polymerase I. Extensive degradation also depends on the early ATP-dependent incision step, and the subsequent degradation shows a partial requirement for ATP. Curtailment of degradation by ligase requires DNA polymerase activity, but is not dependent upon DNA polymerase I. Apparently this process can be carried out with equal facility by either DNA polymerase II or polymerase III. These observations suggest that extensive DNA polymerase I-dependent repair synthesis and extensive DNA degradation are facets of two divergent pathways of excision repair, both of which depend upon the early uvrABC determined ATP-dependent incision step. (orig.) [de

  7. 67Ga-citrate incorporation and DNA synthesis in tumors

    International Nuclear Information System (INIS)

    Hammersley, P.A.G.; Taylor, D.M.

    1975-01-01

    The results obtained in these studies suggest that in the tumors studied there is some form of relationship between 67 Ga uptake and the rate of DNA synthesis. However, the observations in the HP melanoma, in which small tumors showed a negative correlation between 67 Ga uptake and rate of DNA synthesis and larger tumors showed a positive correlation, coupled with the virtually constant uptake of 67 Ga over a wide range of rates of DNA synthesis in the drug- and radiation-treated tumors, suggest that the uptake of the radionuclide is not simply related to the rate of DNA synthesis per se. Studies in embryonic mouse tissues suggested that 67 Ga uptake was not related to the rate of DNA synthesis and regenerating liver does not show a greater 67 Ga uptake than normal liver. Phytohemagglutinin-treated human lymphocytes show increased 67 Ga uptake compared to unstimulated lymphocytes, and it has been suggested that this is related to the stimulus to divide rather than to events occurring in a specific phase of the cell cycle. This suggests that proliferating cells may exhibit membrane changes which either result in increased transport of 67 Ga into the cell or permit a greater degree of binding of the radionuclide to the cell membrane than can occur in resting cells. The membrane-binding hypothesis is supported by the observations on phytohemagglutinin-stimulated lymphocytes but not by observations of the subcellular distribution of 67 Ga in these tumors which confirm the suggestion that the radionuclide is concentrated in lysosomes. Thus it appears that although in tumor cells, at least, there is some correlation between 67 Ga uptake and the rate of DNA synthesis and hence by implication of cell proliferation, the nature of this link remains obscure, and more detailed studies are needed to increase our understanding of the relationship

  8. ATM Protein Physically and Functionally Interacts with Proliferating Cell Nuclear Antigen to Regulate DNA Synthesis*

    Science.gov (United States)

    Gamper, Armin M.; Choi, Serah; Matsumoto, Yoshihiro; Banerjee, Dibyendu; Tomkinson, Alan E.; Bakkenist, Christopher J.

    2012-01-01

    Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner. PMID:22362778

  9. Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation

    International Nuclear Information System (INIS)

    Ventura, A.M.; Meneghini, R.

    1984-01-01

    Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nonsubstituted DNA, was noticed in the density-distribution profiles. The density distribution of DNA along the gradient can provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement. (Auth.)

  10. Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

    International Nuclear Information System (INIS)

    Ulrich, F.

    1988-01-01

    When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [ 3 H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others,suggest that deoxyribose damages DNA

  11. Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

    International Nuclear Information System (INIS)

    Dresler, S.L.; Gowans, B.J.; Robinson-Hill, R.M.; Hunting, D.J.

    1988-01-01

    DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

  12. Effect of inhibition of DNA synthesis on recovery of X-irradiated L5178Y-S cells. I

    International Nuclear Information System (INIS)

    Kapiszewska, M.; Lange, C.S.

    1989-01-01

    Irradiated L5178Y-S cells (LY-S) were characterized by an exponential survival curve and the potentiation effect of split -dose irradiation. Previously it was found that in LY-S cells the reduction of DNA replicative synthesis rate affected the balance between the fixation and repair of sublethal damage (SLD) and of potentially lethal damage (PLD) in favor of repair. It was found now that a block of DNA synthesis by aphidicolin (APC), an inhibitor of DNA polymerase alpha, was sufficient to protect LY-S cells from fixation of PLD and SLD induced by X-rays. Treatment with APC 0.5 μg/ml for 2 h, efficiently inhibited DNA replication (95%) with minimal effect on survival. Inhibition of DNA synthesis by combined irradiation and APC was not significantly different from APC treatment alone. The level of protection by APC was dependent on the length of time between irradiation and APC application. An opposite effect was observed when the drug treatment had preceded irradiation: The killing effect of X-ray increased. The effect of aphidicolin treatment remained even after removal of APC and was dependent on the drug concentration and time between drug removal and irradiaton. These results are interpreted as indicating that X-ray damage was fixed in LY-S cells, because of their lack of ability to maintain the nucleotide pool balance, and that fixation took place during progression through the cell cycle. (author). 6 figs., 22 refs

  13. Inhibition and recovery of semiconservative DNA synthesis in normal and solar UV sensitive ICR 2A frog cell lines following the induction of non-dimer DNA damage by sunlamp UV > 315 nm

    Energy Technology Data Exchange (ETDEWEB)

    Rosenstein, B.S. (Brown Univ., Providence, RI (USA). Dept. of Radiation Medicine)

    1989-08-01

    Cultures of solar UV-sensitive cell lines DRP 36 and DRP 153, and of the parental ICR 2A cell line, were exposed to 150 kJ/m{sup 2} of sunlamp UV>315nm plus photoreactivating light, resulting in the induction primarily of non-dimer DNA damage. Following either 0, 3, 6, 12 or 24 h incubation, cultures were pulse-labelled with ({sup 3}H) thymidine, and the synthesis of different size classes of replicon intermediates measured using alkaline step elution assay. For all three cell lines tested, an immediate depression of low molecular weight DNA synthesis was observed, followed by inhibition of all size classes of replicon intermediates. Within 12 h following irradiation, recovery of DNA synthesis was observed, generally most apparent for low molecular weight DNA. The ICR 2A cells exhibited a nearly full recovery in all size classes of DNA synthesized by 24 h. A much smaller recovery of continued inhibition was primarily in the synthesis of full replicon size DNA, and most pronounced for DRP 36 cells. (author).

  14. Age-dependence of the x-ray-induced deficiency in DNA synthesis in HeLa S3 cells during generation 1

    International Nuclear Information System (INIS)

    Griffiths, T.D.; Tolmach, L.J.

    1975-01-01

    The radiation-induced deficiency in DNA synthesis in Generation 1 was studied as a function of the age of HeLa S3 cells at the time of exposure to 220 kV x rays in the previous generation (Generation 0). The amount of DNA synthesized is dependent on the stage in the generation cycle at which cells are irradiated. The smallest deficiency (20 to 35 percent after a dose of 500 rad) is observed in cells irradiated in early G1 or early G2, while the greatest deficiency (55 to 70 percent after 500 rad) is found in cells irradiated at mitosis or at the G1/S transition. The high sensitivity of cells at G1/S is also manifested by a steeper dose-response curve. Cells irradiated in late G2, past the point where their progression is temporarily blocked by x rays, synthesize a normal amount of DNA in Generation 1, while cells that are held up in the G2 block exhibit deficient synthesis in the next generation. The extent of the deficiency in early G1 cells can be enhanced by treatment with 1 mM hydroxyurea for several hours immediately following irradiation. The possibility that deficient DNA synthesis is related to cell killing, and the relation between the G2 block and deficient synthesis, are discussed

  15. DNA-membrane complex restoration in Micrococcus radiodurans after X-irradiation: relation to repair, DNA synthesis and DNA degradation

    Energy Technology Data Exchange (ETDEWEB)

    Dardalhon-Samsonoff, M; Averbeck, D [Institut du Radium, 75 - Paris (France). Lab. Curie

    1980-07-01

    The DNA-membrane complex in Micrococcus radiodurans was shown to be essentially constituted of proteins, lipids and DNA. The complex was dissociated immediately after X-irradiation of cells and restored during post-incubation in complete medium. In X-irradiated protoplasts some DNA remained associated with the complex. Restoration of the complex during post-incubation was only seen in a medium favouring DNA polymerase and ligase activities. Under this condition no DNA synthesis occurred, suggesting that complex restoration may involve ligase activity. The complex restoration in the wild type and the X-ray sensitive mutant UV17 of M. radiodurans was strictly dependent on the X-ray dose. It was correlated with survival and DNA degradation but always preceded the onset of DNA synthesis after X-irradiation. At the same dose the complex restoration was about 2 fold lower in mutant than in wild type cells indicating that the restoration of the complex is related to repair capacity. The results are consistent with the idea that the complex protects X-irradiated DNA of M. radiodurans from further breakdown and, subsequently, permits DNA synthesis and repair to occur.

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

    International Nuclear Information System (INIS)

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

    1991-01-01

    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 [ 3 H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [ 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 [ 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

  17. Radioresistant DNA synthesis in fibroblasts of a patient with Down's syndrome

    International Nuclear Information System (INIS)

    Barenfel'd, L.S.; Bil'din, V.N.; Pleskach, N.M.; Prokof'eva, V.V.

    1985-01-01

    Ionizing radiation effect on DNA replication on fibroblasts of a healthy donor and a patient with Down's syndrome either by direct 3 H-thymidine inclusion into DNA, or by analysis of the sizes of daughter DNA moleculs at the state of stable distribution in acid saccharose, gradients was studied. Gamma-radiation doses (5-10 Gy) suppressing DNA synthesis in normal fibroblasts practically had no effect on DNA synthesisin fibroblasts of a patient with Down's syndrome. Radioresistant DNA synthesis in Down's syndrome is conditioned by a far less supression of replicon initiation as compared with the one in normal cells. So, it is stated that in Down's disease there is no delay in DNA synthesis by ionizing radiation that enables the normal cells to repair DNA damages before replication renewal

  18. DNA replication and repair of Tilapia cells: Pt. 2

    International Nuclear Information System (INIS)

    Chen, J.D.; Yew, F.H.

    1988-01-01

    TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34 0 C). We report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 0 C to the sublethal high temperature of 37 0 C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37 0 C the rate of DNA synthesis overshot the 31 0 C control level by 180%. When moved to low temperature (18 0 C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. U.V. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3h at 37, 31 and 25 0 C, but not at 18 0 C. Initiation of nascent DNA synthesis was blocked at 4Jm -2 in TO-2 cells compared with ≤ 1Jm -2 in mammalian cells. After 9Jm -2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate. TO-2 cells showed low levels of u.v.-induced excision repair. (author)

  19. Action of some drugs on enzymes involved in DNA-repair and semiconservative DNA-synthesis

    International Nuclear Information System (INIS)

    Wawra, E.; Klein, W.; Kocsis, F.; Weniger, P.

    1975-07-01

    Different antirheumatic and cytostatic drugs had been tested by measurement of the thymidine incorporation into DNA of spleen cells under conditions, under which either DNA-synthesis or repair after gamma- or UV-irradiation takes place. There are substances, which inhibit either only the semiconservative DNA-synthesis (vinblastine, isonicotinic acid hydracide) or only DNA-repair after gamma-irradiation (mixture of penicillin-G and procaine-penicillin-G) or both (cyclophosphamide, phenylbutazone, procarbazine, nalidixic acid). Vincristine shows no effect on the thymidine incorporation in DNA, but by density gradient centrifugation it has been found that it influences the ligase reaction. Two DNA polymerases had been isolated from spleen cells, one of the low molecular and one of the high molecular weight type. The influences of the described drugs on these enzymes and on a deoxyribonuclease I from beef pancreas have been tested in ''in vitro'' systems. In all cases, it has been found that there is no effect or only a very small one, compared with the action of well known inhibitors as e.g. ethidium bromide and p-chloromercuribenzoate, and this cannot be responsible for the suppressions found in DNA-repair and semiconservative DNA-synthesis. (author)

  20. Influence of some radioprotective and radiosensitizing compounds on the replicative and repair induced DNA synthesis of rats spleen cells in vitro

    International Nuclear Information System (INIS)

    Goette, A.

    1982-01-01

    The effect of cysteine, dithiothreitol, N-ethylmaleimide, cytosinearabinoside, ethidiumbromide, bleomycine and diethyldithiocarbamate on the replicative and repair induced DNA synthesis in vitro was tested by using rats spleen cells. Besides the incorporation of a labeled DNA precursor (TdR- 3 H) the sedimentation of DNA in sucrose gradients was inquired. With respect to the DNA synthesis an uniform mechanism of action for the radioprotective substances can't be seen. Thymocytes and spleen cells seem to possess different systems of repair; this may be an explanation for their different sensibility against ionizing radiation. (orig./MG) [de

  1. DNA replication and repair of Tilapia cells: Pt. 2. Effects of temperature on DNA replication and ultraviolet repair in Tilapia ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.D.; Yew, F.H.

    1988-02-01

    TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34/sup 0/C). We report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31/sup 0/C to the sublethal high temperature of 37/sup 0/C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37/sup 0/C the rate of DNA synthesis overshot the 31/sup 0/C control level by 180%. When moved to low temperature (18/sup 0/C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. U.V. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3h at 37, 31 and 25/sup 0/C, but not at 18/sup 0/C. Initiation of nascent DNA synthesis was blocked at 4Jm/sup -2/ in TO-2 cells compared with less than or equal to 1Jm/sup -2/ in mammalian cells. After 9Jm/sup -2/ u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate. TO-2 cells showed low levels of u.v.-induced excision repair.

  2. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Hammond, R.A.; Miller, M.R.; McClung, J.K.

    1990-01-01

    The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

  3. DNA replication and repair in Tilapia cells

    International Nuclear Information System (INIS)

    Yew, F.H.; Chang, L.M.

    1984-01-01

    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-β-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor. (author)

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

    Science.gov (United States)

    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)

  5. Estrogen-induced DNA synthesis in vascular endothelial cells is mediated by ROS signaling

    Directory of Open Access Journals (Sweden)

    Felty Quentin

    2006-04-01

    Full Text Available Abstract Background Since estrogen is known to increase vascular endothelial cell growth, elevated estrogen exposure from hormone replacement therapy or oral contraceptives has the potential to contribute in the development of abnormal proliferative vascular lesions and subsequent thickening of the vasculature. How estrogen may support or promote vascular lesions is not clear. We have examined in this study whether estrogen exposure to vascular endothelial cells increase the formation of reactive oxygen species (ROS, and estrogen-induced ROS is involved in the growth of endothelial cells. Methods The effect of estrogen on the production of intracellular oxidants and the role of estrogen-induced ROS on cell growth was studied in human umbilical vein endothelial cells. ROS were measured by monitoring the oxidation of 2'7'-dichlorofluorescin by spectrofluorometry. Endothelial cell growth was measured by a colorimetric immunoassay based on BrdU incorporation into DNA. Results Physiological concentrations of estrogen (367 fmol and 3.67 pmol triggered a rapid 2-fold increase in intracellular oxidants in endothelial cells. E2-induced ROS formation was inhibited to basal levels by cotreatment with the mitochondrial inhibitor rotenone (2 μM and xanthine oxidase inhibitor allopurinol (50 μM. Inhibitors of NAD(PH oxidase, apocynin and DPI, did not block E2-induced ROS formation. Furthermore, the NOS inhibitor, L-NAME, did not prevent the increase in E2-induced ROS. These findings indicate both mitochondria and xanthine oxidase are the source of ROS in estrogen treated vascular endothelial cells. E2 treated cells showed a 2-fold induction of BrdU incorporation at 18 h which was not observed in cells exposed to vehicle alone. Cotreatment with ebselen (20 μM and NAC (1 mM inhibited E2-induced BrdU incorporation without affecting the basal levels of DNA synthesis. The observed inhibitory effect of NAC and ebselen on E2-induced DNA synthesis was also shown

  6. UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein

    International Nuclear Information System (INIS)

    Carty, M.P.; Zernik-Kobak, M.; McGrath, S.; Dixon, K.

    1994-01-01

    We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus. (Author)

  7. Differential responses of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to U.V. light and chemical mutagens

    International Nuclear Information System (INIS)

    Fox, M.; Bloomfield, M.E.; Hopkins, J.; Boyle, J.M.

    1983-01-01

    DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethane sulphonate (EMS) in Chinese hamster V79 cells and the mutagen sensitive derivative V79/79 was investigated by measurement of five parameters: production of strand breaks in template DNA, incorporation of [ 3 H]TdR, semi-conservative and repair synthesis, molecular weights of pulse labelled DNA after mutagen exposure (nascent synthesis) and molecular weights of DNA pulse labelled and chased after mutagen exposure (elongation and ligation). Equal template strand breakage was evident in both cell lines immediately after MNU and EMS exposure and by 4-5 h after MNU the extent of fragmentation was greater in V79/79 cells. After u.v. irradiation template fragmentation was evident in V79/79 but not in V79 cells, even though V79/79 cells failed to excise cyclobutane dimers and repair synthesis was demonstrable in V79 cells but not in V79/79 cells after exposure to all three mutagens. The rate of incorporation of [ 3 H]TdR during semi-conservative DNA synthesis was inhibited equally in a dose dependent manner after u.v. and MNU exposure; incorporation by V79/79 cells was inhibited to a greater extent than by V79 cells after EMS exposure. Nascent DNA synthesis was suppressed more in V79/79 cells than in V79 cells after u.v. but to similar extents in both cell lines after MNU and EMS treatment. Pulse chase experiments indicated a lower rate of elongation of nascent DNA in V79/79 cells after MNU and u.v. exposure but little difference was detectable after EMS

  8. The impact of cofactors and inhibitors on DNA repair synthesis after γ-irradiation in semi-permeable Escherichia coli cells

    International Nuclear Information System (INIS)

    Gaertner, C.

    1981-01-01

    The DNA-repair synthesis in tuluol-permeable E. coli cells after γ-irradiation has been investigated in dependence on the co-facotrs. ATB and NAD by means of enzyme kinetics. A partly repair-deficient mutants were taken into consideration which are well characterized in view of molecular biology; they showed which enzyme functions participate in the γ-induced DNA repair synthesis. The inhibition of the DNA-repair synthesis by the intercalary substances Adriamycin and Proflavin has been described and compared with the survival rates after irradiation and after combined treatment by irradiation and intercalary agents. (orig./AJ) [de

  9. Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: An indication of cell cycle defects

    International Nuclear Information System (INIS)

    Hannan, Mohammed A.; Kunhi, Mohammed; Einspenner, Michael; Khan, Bashir A.; Al-Sedairy, Sultan

    1994-01-01

    In the present study, both post-irradiation DNA synthesis and G 2 phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkin's lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by 3 H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G 2 phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G 2 phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G 2 accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cycle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G 2 phase accumulation developed elsewhere in characterizing AT heterozygote-like cell cycle anomaly in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle

  10. Unscheduled DNA synthesis in spleen cells of mice exposed to low doses of total body irradiation

    International Nuclear Information System (INIS)

    Tuschl, H.; Kovac, R.; Hruby, E.

    1983-07-01

    Unscheduled DNA synthesis was induced by UV irradiation of spleen cells obtained from C 57 Bl mice after repeated total body irradiation of 0.05 Gy 60 Co (0.00125 Gy/mice) and determined autoradiographically. An enhancement in the ability for repair of UV induced DNA lesions was observed in cells of gamma irradiated animals. While the amount of 3 H-thymidine incorporated per cell was increased, the percentage of labeled cells remained unchanged. The present results are compared with previous data on low dose radiation exposure in men. (Author) [de

  11. Replication stress activates DNA repair synthesis in mitosis

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A

    2015-01-01

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps...... into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early...... mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...

  12. Abnormal levels of UV-induced unscheduled DNA synthesis in ataxia telangiectasia cells after exposure to ionizing radiation

    International Nuclear Information System (INIS)

    Jaspers, N.G.J.; Nederlandse Centrale Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek, Rijswijk. Medical Biological Lab.); Bootsma, D.

    1982-01-01

    In cultured cells from normal individuals and from patients having ataxia telangiectasia (AT) the rate of unscheduled DNA synthesis (UDS) induced by UV light was investigated by autoradiography. The number of grains in 6 different AT cell strains was similar to that observed in normal cells. Exposure of normal cells to doses of X-rays up to 20 krad had no influence on the rate of UV-induced UDS. In contrast, the UV-induced UDS was significantly modified in AT cells by treatment with X-rays. In AT cell strains that were reported to have reduced levels of γ-ray-induced repair DNA synthesis ('excision-deficient' AT cells) the effect of X-rays on UV-induced UDS was inhibitory, whereas UV-induced UDS was stimulated by X-ray exposure in 'excision-proficient' AT cell strains. Different UV and X-ray dose-response relationships were seen in the two categories of AT cell strains. (orig./AJ)

  13. Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

    International Nuclear Information System (INIS)

    Park, S.D.; Cleaver, J.E.

    1979-01-01

    Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [ 3 H] thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rated of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. These observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair. (author)

  14. Comparative investigations about the DNA synthesis by thymus and spleen cells of rats in vitro under the influence of X-rays, UV radiation and radiomimetic substances

    Energy Technology Data Exchange (ETDEWEB)

    Tempel, K.; Schmerold, I.; Pfahler, W.; Goette, A.

    1984-06-01

    In order to further characterize the different repairing behavior of thymus and spleen cells of rats in vitro under the influence of X-rays, UV radiation and methylmethanesulfonate (MMS), the effect of bleomycine (BM), L-cysteine (CY-E), N-ethylmaleimide (NEM), I-..beta..-D-arabinofuranosylcytosine (araC), dideoxythymidine (ddT), and novobiocine (NB) on the semiconservative and restorative DNA synthesis as well as on the behavior of DNA under the alkaline elution was studied. The semiconservative DNA synthesis was inhibited by all examined agents except ddT, the restorative DNA synthesis only by NEM, araC, and NB. The stimulation of the restorative DNA synthesis was increased by UV radiation and MMS in spleen cells and by X-rays, BM and CY-E in thymus cells. Under the conditions of alkaline elution, there was a more sensitive reaction of spleen cells than of thymus cells to X-rays, BM and CY-E. The results show that thymus cells are especially qualified for the repair of short chains and spleen cells for the repair of long chains.

  15. DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.

    Science.gov (United States)

    Ohmine, Yuta; Satoh, Yukari; Kiyokawa, Kazuya; Yamamoto, Shinji; Moriguchi, Kazuki; Suzuki, Katsunori

    2016-04-02

    Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome. By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence. RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high

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

    DEFF Research Database (Denmark)

    Levring, Trine B; Kongsbak-Wismann, Martin; Rode, Anna Kathrine Obelitz

    2015-01-01

    . 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 Cys......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...... 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...

  17. Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

    International Nuclear Information System (INIS)

    Nishiyama, Y.; Rapp, F.

    1981-01-01

    Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants

  18. Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

    International Nuclear Information System (INIS)

    Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

    1986-01-01

    The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [ 3 H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [ 3 H]thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density

  19. RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

    Science.gov (United States)

    Yu, W; Sanders, B G; Kline, K

    1997-01-01

    RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

  20. Continuous induction of unscheduled DNA synthesis by gamma irradiation

    International Nuclear Information System (INIS)

    Weniger, P.; Klein, W.; Ott, E.; Kocsis, F.; Altmann, H.

    1990-01-01

    The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA. Usually, in an in vivo - in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process - with centrifugation in alkaline sucrose a half-life of some minutes is found - an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation. (author) 4 figs

  1. Continuous induction of unscheduled DNA synthesis by gamma irradiation

    International Nuclear Information System (INIS)

    Weniger, P.; Klein, W.; Ott, E.; Kocsis, F.; Altmann, H.

    1988-08-01

    The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of the DNA. Usually, in an in vivo -in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation should be observed for a long time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten points of time during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process - with centrifugation in alkaline sucrose you find a half time of some minutes - an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation. 4 figs. (Author)

  2. DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients.

    Science.gov (United States)

    Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

    2009-07-14

    Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

  3. DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

    Science.gov (United States)

    Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

    2009-01-01

    Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load. PMID:19564618

  4. DNA repair and DNA synthesis in leukemic and virus infected cells

    International Nuclear Information System (INIS)

    Tuschl, H.; Altmann, H.; Kovac, R.; Topaloglou, A.; Stacher, A.; Fanta, D.

    1978-09-01

    Autoradiographic determinations of unscheduled DNA synthesis in peripheral lymphocytes of leukemic patients showed strongly different results according to various types of disease of different forms of therapy, respectively. Similar investigations performed with lymphocytes of Herpes simplex infected persons during symptom-free intervals revealed imbalances of the repair system caused by virus infection. BND cellulose chromatography and measurement of 3 H-thymidine incorporation into single- and double stranded DNA fractions showed an increase in velocity of the rejoining process, but a decrease in total incorporation. Because of these results and the demonstration of the supercoiled structure of DNA it is suggested that virusinfections cause a faster rejoining of gaps, but at the same time leave a number of failures within DNA unrecognized. (author)

  5. Histone gene expression remains coupled to DNA synthesis during in vitro cellular senescence

    International Nuclear Information System (INIS)

    Zambetti, G.; Stein, G.; Stein, J.; Dell'Orco, R.

    1987-01-01

    Despite a decrease in the extent to which confluent monolayers of late compared to early passage CF3 human diploid fibroblasts can be stimulated to proliferate, the time course of DNA synthesis onset is similar regardless of the in vitro age of the cells. A parallel and stoichiometric relationship is maintained between the rate of DNA synthesis and the cellular levels of histone mRNA independent of the age of the cell cultures. Furthermore, DNA synthesis and cellular histone mRNA levels decline in a coordinate manner after inhibition of DNA replication by hydroxyurea treatment. These results indicate that while the proliferative activity of human diploid fibroblasts decreases with passage in culture, those cells that retain the ability to proliferate continue to exhibit a tight coupling of DNA replication and histone gene expression

  6. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Tetsuya, E-mail: suzukite@hiroshima-u.ac.jp [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Grúz, Petr; Honma, Masamitsu [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan); Adachi, Noritaka [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027 (Japan); Nohmi, Takehiko [Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501 (Japan)

    2016-09-15

    Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

  7. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

    International Nuclear Information System (INIS)

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    2016-01-01

    Highlights: • Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells. • Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments. • Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links. • Pol ζ plays quite limited roles in protection against strand-breaks in DNA. - Abstract: Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were

  8. Impairment of DNA synthesis in Gunn rat cerebellum.

    Science.gov (United States)

    Yamada, N; Sawasaki, Y; Nakajima, H

    1977-05-06

    Brain DNA synthesis was developmentally investigated in Gunn rat with marked cerebellar hypoplasia due to hereditary hyperbilirubinemia. In this mutant rat, the Purkinje cell was nearly selectively affected in the cerebellar cortex by bilirubin. The impaired DNA synthesis was observed in homozygous (jj) Gunn rat cerebellum, in which the DNA content and [3H]thymidine incorporation rate into DNA decreased after 10 days of age compared to those in the heterozygous (Jj)littermate. In contrast, these impairments were not found in the non-cerebellar parts of the brain and liver of jj Gunn rat. The activity of cerebellar thymidine kinase in jj Gunn rat decreased from a very early stae, being 80% of Jj rat at 6 days, and 50% at 10 days of age. The enzyme activity was not affected in the non-cerebellar parts of the brain. Although bilirubin competitively inhibited cerebellar thymidine kinase activity in vitro (15% at 10(-5) M), such bilirubin level was found to be about 1000-fold that in vivo. Moreover, photo-degradation of bilirubin in jj cerebellum exhibited no improvement in thymidine kinase activity, and the presence of an enzyme inactivator was not suggested in jj cerebellum. These results seem to indicate that the induction of thymidine kinase might be affected in jj Gunn rat cerebellum. The possibility that the impaired DNA synthesis in the external granular cells in jj cerebellum may be due to Purkinje cell damage is discussed.

  9. Evidence that transferrin supports cell proliferation by supplying iron for DNA synthesis

    International Nuclear Information System (INIS)

    Laskey, J.; Webb, I.; Schulman, H.M.; Ponka, P.

    1988-01-01

    Transferrin is essential for cell proliferation and it was suggested that it may trigger a proliferative response following its interaction with receptors, serving as a growth factor. However, since the only clearly defined function of transferrin is iron transport, it may merely serve as an iron donor. To further clarify this issue, the authors took advantage of an iron chelate, ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH), which they developed and previously demonstrated to efficiently supply iron to cells without using physiological transferrin receptor pathway. As expected, they observed that blocking monoclonal antibodies against transferrin receptors inhibited proliferation of both Raji and murine erythroleukemia cells. This inhibited cell growth was rescued upon the addition of Fe-SIH which was also shown to deliver iron to Raji cells in the presence of blocking anti-transferrin receptor antibodies. Moreover, blocking anti-transferrin receptor antibodies inhibited [ 3 H]thymidine incorporation into DNA and this inhibition could be overcome by added Fe-SIH. In addition, Fe-SIH slightly stimulated, while SIH (an iron chelator) significantly inhibited, DNA synthesis in phytohemagglutinin-stimulated peripheral blood lymphocytes. Taken together, these results indicate that the only function of transferrin supporting cell proliferation is to supply cells with iron

  10. Effect of UV on DNA synthesis in UV-resistant insect cells

    International Nuclear Information System (INIS)

    Styer, S.C.; Meechan, P.J.; Griffiths, T.D.

    1987-01-01

    Insect cells are most resistant to killing by 254 nm ultraviolet light (UV) than mammalian cells. Because they have an active photolyase, it may be possible to generate a higher number of [6-4] PyC lesions per genome, allowing the possibility to distinguish between the effects of [5-6] pyrimidine lesions and the nonphotoreactable [6-4] lesions on DNA replication. IAL-PID2 cells, derived from imaginal wing discs of the Indian meal moth were exposed to UV followed by photoreactivating light (PR) or sham treatment and then analyzed by measuring the incorporation of [/sup 3/H]-thymidine into acid precipitable form. As expected, there was a fluence-dependent decrease in the amount of thymidine incorporated after exposure to UV. The response was similar to that observed in wild type CHO cells (AAS) except that the rate of decline was more rapid. When PR followed UV, there was less of a decline in thymidine incorporation and a more rapid recovery. However, thymidine incorporation did not return to control levels as rapidly as expected if [5-6] lesions were the only lesions involved in the disruption of DNA synthesis after exposure to UV

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

    International Nuclear Information System (INIS)

    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

  12. DNA synthesis in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Painter, R.B.; California Univ., San Francisco; Young, B.R.

    1987-01-01

    One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v. -induced inhibition of replicon initiation. (author

  13. Normal inhibition of DNA synthesis following γ-irradiation of radiosensitive cell lines from patients with Down's syndrome and Alzheimer's disease

    International Nuclear Information System (INIS)

    Lavin, M.F.; Le poidevin, P.; Chen, P.C.; Bates, P.

    1989-01-01

    Inhibition of DNA synthesis was studied in γ-iradiated lymphoblastoid cells from patients with Alzheimer's disease and Down's syndrome. A normal biphasic pattern of inhibition was observed over a dose range of 0-4 krad of γ-rays in all of the cell lines 3 out of 4 Down's and all the Alzheimer's cell lines were shown to be hypersensitive to ionizing radiation based on induced chromosomal aberrations. Increased G2 phase delay, comparable to that occurring in ataxia-telangiectasia cells, was observed for some of the cell lines, after exposure to γ-rays. Contrary to other data in the literature these results demonstrate that radioresistand DNA synthesis is not an intrinsic feature of all disorders characterized by radiosensitivitey. (author).; 25 refs.; 2 figs.; 1 tab

  14. Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

    Science.gov (United States)

    Gan, Haiyun; Yu, Chuanhe; Devbhandari, Sujan; Sharma, Sushma; Han, Junhong; Chabes, Andrei; Remus, Dirk; Zhang, Zhiguo

    2017-10-19

    The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli

    International Nuclear Information System (INIS)

    Dorson, J.W.; Moses, R.E.

    1978-01-01

    DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' yields 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair

  16. Nucleic acids synthesis of nuclear polyhedrosis virus in cultured embryonic cells of silkworm

    International Nuclear Information System (INIS)

    Himeno, Michio; Kimura, Yukio; Hayashiya, Keizo.

    1976-01-01

    Embryos of the silkworm, Bombyx mori L., were dispersed by trypsin and the dissociated cells were cultured for infection with nuclear polyhedrosis virus (NPV) of the silkworm. The monolayer and suspension cultures were infected with NPV. RNA and DNA syntheses in the normal and NPV-infected cells were measured by incorporation of 32 P into RNA and DNA fractions. RNA and DNA syntheses in the cells after infection significantly increased over those in control cells (mock infection). The effects of actinomycin D, chloramphenicol and mitomycin C on RNA and DNA syntheses in infected cells were examined. The syntheses were inhibited by the antibiotics. It was suggested that the cellular DNA synthesis was inhibited by the viral infection, because the mitomycin C-resistant DNA synthesis was found in the normal cells but not in the infected cells treated with mitomycin C. The rate of DNA synthesis induced by NPV was immediately dropped to that of control cells by addition of chloramphenicol, while the RNA synthesis induced by NPV was not affected for 6 hr after the addition of chloramphenicol. If the antibiotic did not affected the size of precursor pools, this event suggested that the RNA polymerase concerned with viral RNA synthesis was more stable than the DNA polymerase participating in the viral DNA synthesis. The viral DNA as templates for RNA and DNA syntheses was decomposed by mitomycin C. (auth.)

  17. Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

    Science.gov (United States)

    Makarova, A V; Kulbachinskiy, A V

    2012-06-01

    Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

  18. The effect of purine phosphonomethoxyalkyl derivatives on DNA synthesis in Cho Chinese hamster cells

    Energy Technology Data Exchange (ETDEWEB)

    Stetina, R [Institute of Experimental Medicine, Laboratory of Developmental Toxicology, Academy of Sciences of Czech Republic, 51783 Olesnice v Orlickych horach (Czech Republic); Votruba, I; Holy, A; Merta, A [Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic (Czech Republic)

    1994-12-31

    The inhibition of incorporation of {sup 3}H-thymidine and the changes of the rate of nascent DNA chain elongation were investigated in Cho Chinese hamster cells treated with (S)-(3-hydroxy-2-phosphonomethoxypropyl) (HPMP) and N-(2-phosphonomethoxyethyl) (PME) derivatives of adenine (A), guanine (G) and 2,6-diaminopurine (DAP). No direct correlation was observed in PME and HPMP derivatives between cytotoxicity, inhibition of {sup 3}H-thymidine incorporation and inhibition of nascent DNA chain elongation. The highest cytotoxicity and inhibition of DNA synthesis were caused by PMEG. The limited extent of inhibition of DNA elongation was encountered in the case of HPMPG and HPMPA. With PMEA, weak inhibition of elongation of DNA was observed only after a prolonged exposure (6 h). None of the investigated drugs induced DNA breaks. (author) 4 figs., 23 refs.

  19. Induction of unscheduled DNA synthesis on the nuclear matrix of rat hepatocytes after whole-body γ-irradiation

    International Nuclear Information System (INIS)

    Bezlepkin, V.G.; Malinovskij, Yu.Yu.; Kuznetsova, E.A.; Namvar, R.A.; Gaziev, A.I.

    1986-01-01

    DNA synthesis in hepatocytes was studied by incorporation of [ 3 H]thymidine administered of portal vein of γ-irradiated (80 Gy) rats. It was shown that the rate of replicative DNA synthesis decreased in hepatocytes of the regenerating liver and unscheduled DNA synthesis was induced at the nuclear matrix of resting cells of the intact liver. In addition to repair synthesis, DNA synthesis resembling replicative one (''aberrant'' DNA synthesis) accounts for a considerable fraction of γ-radiation-induced synthesis of DNA at the nuclear matrix

  20. Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine.

    Science.gov (United States)

    Yoon, Jung-Hoon; Roy Choudhury, Jayati; Park, Jeseong; Prakash, Satya; Prakash, Louise

    2017-11-10

    N3-Methyladenine (3-MeA) is formed in DNA by reaction with S -adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro , we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

    Science.gov (United States)

    van Erp, Y H; Koopmans, M J; Heirbaut, P R; van der Hoeven, J C; Weterings, P J

    1992-06-01

    A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.

  2. DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

    Directory of Open Access Journals (Sweden)

    Yaofeng Li

    2015-03-01

    Full Text Available 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.

  3. Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis

    International Nuclear Information System (INIS)

    Ganges, M.B.; Robbins, J.H.; Jiang, H.; Hauser, C.; Tarone, R.E.

    1988-01-01

    The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radioresistant DNA synthesis. (author). 27 refs.; 1 fig.; 1 tab

  4. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  5. Deficient repair of chemical adducts in alpha DNA of monkey cells

    International Nuclear Information System (INIS)

    Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

    1982-01-01

    Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

  6. Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C.elegans embryos?

    International Nuclear Information System (INIS)

    Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann

    1991-01-01

    Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs

  7. Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C. elegans embryos

    Energy Technology Data Exchange (ETDEWEB)

    Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann [Texas Christian Univ., Fort Worth, TX (United States). Dept. of Biology

    1991-09-01

    Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs.

  8. Differential response of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to u.v. light and chemical mutagens

    International Nuclear Information System (INIS)

    Fox, M.; Bloomfield, M.E.; Hopkins, J.; Boyle, J.M.

    1983-01-01

    DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethane sulphonate (EMS) in Chinese hamster V79 cells and the mutagen sensitive derivative V79/79 was investigated. Equal template strand breakage was evident in both cell lines immediately after MNU and EMS exposure and by 4-5 h after MNU the extent of fragmentation was greater in V79/79 cells. After u.v. irradiation template fragmentation was evident in V79/79 but not in V79 cells, even though V79/79 cells failed to excise cyclobutane dimers and repair synthesis was demonstrable in V79 cells but not in V79/79 cells after exposure to all three mutagens. The rate of incorporation of [ 3 H]TdR during semi-conservative DNA synthesis was inhibited equally in a dose dependent manner after u.v. and MNU exposure; incorporation by V79/79 cells was inhibited to a greater extent than by V79 cells after EMS exposure. Nascent DNA synthesis was suppressed more in V79/79 cells than in V79 cells after u.v. but to similar extents in both cell lines after MNU and EMS treatment. Pulse chase experiments indicated a lower rate of elongation of nascent DNA in V79/79 cells after MNU and u.v. exposure but little difference was detectable after EMS. (author)

  9. A biochemical defect in the repair of alkylated DNA in cells from an immunodeficient patient (46BR)

    International Nuclear Information System (INIS)

    Teo, I.A.; Broughton, B.C.; Day, R.S.; James, M.R.; Karran, P.; Mayne, L.V.; Lehmann, A.R.

    1983-01-01

    The fibroblast cell strain 46BR, derived from an immunodeficient individual, is hypersensitive to the lethal effects of a variety of DNA-damaging agents, this effect being particularly marked for monofunctional methylating agents. After U.V. irradiation 46BR cells show normal unscheduled DNA synthesis, daughter strand repair, and recovery of DNA and RNA synthesis. The inhibition of DNA replicative synthesis by U.V. is slightly less than that of normal cells. After gamma-irradiation the rejoining of strand breaks is normal as are the kinetics of replicative DNA synthesis. Following treatment with dimethylsulphate, replicative DNA synthesis is affected in a similar way to normal cells, unscheduled DNA synthesis may be increased relative to normal cells, but more strand breaks persist in 46BR than in normal cells. In addition 46BR cells are hypersensitive to the toxic effects of 3-aminobenzamide, an inhibitor of ADP-ribosyl transferase. This enzyme is involved in the ligation step of repair of alkylation damage. A hypothesis is presented suggesting that 46BR may be defective in DNA ligase I

  10. Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1979-01-01

    Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei were both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brought estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells. (author)

  11. Investigations on the mechanism of DNA excision repair in tissue culture cells

    International Nuclear Information System (INIS)

    Wawra, E.; Dolejs, I.; Ott, E.

    1976-12-01

    Semiconservative DNA- synthesis and repair- synthesis was measured in HeLa cells and spleen cells under different conditions (i.e. different temperatures, addition of p-chloromercuribenzoate or cytosine-arabinoside). In order to obtain more information about the enzymatic background of these steps of DNA metabolism, parallel in vitro experiments were done with two different types of DNA polymerase, which had been isolated from pig spleen. At least the experiments at different temperatures are showing some correlations of α-polymerase with semiconservative synthesis and of β-polymerase with repair synthesis. (author)

  12. Typical xeroderma pigmentosum complementation group A fibroblasts have detectable ultraviolet light-induced unscheduled DNA synthesis

    International Nuclear Information System (INIS)

    Petinga, R.A.; Andrews, A.D.; Robbins, J.H.; Tarone, R.E.

    1977-01-01

    Ultraviolet-induced nuclear uptake of tritiated thymidine [ 3 H]dThd demonstrable by autoradiography in non-synthesis phases of the cell cycle is known as unscheduled DNA synthesis and reflects repair replication of ultraviolet-damaged DNA. We have reported that the rate of any such unscheduled DNA synthesis in typical group A xeroderma pigmentosum fibroblasts, if present, is less than 2% of the normal rate. We have now performed experiments to determine whether these fibroblasts have any unscheduled DNA synthesis. Fibroblast coverslip cultures of four xeroderma pigmentosum group A strains were prepared. Irradiated (254 nm ultraviolet light) and unirradiated cultures from each strain were incubated with [ 3 H]dThd at 37degC, and autoradiograms were prepared using NTB-3 emulsion. A nuclear grain count was made of 100 consecutive nuclei of non-S-phase irradiated and unirradiated cells. A slide background grain count was simultaneously made from an acellular area adjacent to each cell analyzed. When a strain's irradiated and unirradiated autoradiograms having similar slide background grain count averages were compared, the nuclear grain count average of the irradiated cells was always higher than that of the unirradiated cells. This ultraviolet-induced increase in the mean nuclear grain count ranged from 0.4 to 1.3% of that given by normal non-xeroderma pigmentosum fibroblasts and was not reduced by 10 -2 M hydroxyurea. Planimetric studies showed that the ultraviolet-induced increase in nuclear grain count is not due to an increased nuclear area in irradiated cells. We conclude that these typical group A xeroderma pigmentosum strains perform very low, but detectable, ultraviolet-induced unscheduled DNA synthesis which probably reflects repair replication. We cannot, however, determine if there are significantly different rates of ultraviolet-induced unscheduled DNA synthesis among these ultraviolet strains

  13. Increased DNA-repair in spleen cells of M. Hodgkin

    International Nuclear Information System (INIS)

    Frischauf, H.; Neumann, E.; Howanietz, L.; Dolejs, I.; Tuschl, H.; Altmann, H.

    1974-11-01

    In spleen cells of control patients and cells of Morbus Hodgkin, DNA-repair after gamma- and UV-irradiation was determined measuring the incorporated 3H-thymidine activity in the DNA. Additionally, the ratio of labeled cells compared to non-labeled cells and the grains per cell were evaluated by autoradiographic investigations. DNA-content per cell was measured using pulsecytophotometry. A significant increase of DNA-repair capacity after gamma-irradiation was found by density gradient centrifugation in alkaline sucrose. The same trend could be shown by investigations of unscheduled DNA-synthesis using autoradiographic method. (author)

  14. Variations in DNA synthesis and mitotic indices in hepatocytes and sinusoid litoral cells of adult intact male mouse along a circadian time span.

    Science.gov (United States)

    Surur, J M; Moreno, F R; Badrán, A F; Llanos, J M

    1985-01-01

    Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.

  15. Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1989-01-01

    SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G 2 chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author)

  16. Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells

    International Nuclear Information System (INIS)

    Clark, J.M.; Hanawalt, P.C.

    1982-01-01

    The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with 3 H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA

  17. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    International Nuclear Information System (INIS)

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert; Rozengurt, Enrique

    2013-01-01

    Highlights: ► Metformin inhibits cancer cell growth but the mechanism(s) are not understood. ► We show that the potency of metformin is sharply dependent on glucose in the medium. ► AMPK activation was enhanced in cancer cells incubated in physiological glucose. ► Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. ► 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 79 and Raptor at Ser 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 α 1 and α 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.

  18. Immediate effects of grenz rays on epidermal DNA synthesis in the flanks of guinea pigs

    International Nuclear Information System (INIS)

    Daikeler, G.

    1976-01-01

    The following findings were obtained by autoradiography: 1) Labelling index (number of labelled cell nuclei per 1,000 based cells): Significant decrease immediately after exposure to grenz rays. 2) Silver grain index (number of silver cells as a function of the labelled basal cells): Significant decrease after irradiation. 3) DNA synthesis index (product of labelling index and silver grain index): Sifnificant decrease of the actual DNA synthesis rate of the reproductive cell cluster after exposure to grenz rays. (orig./AJ) [de

  19. Effects of ionizing radiations on DNA replication in cultured mammalian cells

    International Nuclear Information System (INIS)

    Makino, F.; Okada, S.

    1975-01-01

    The dose-response curve of [ 3 H] thymidine incorporation into the acid-insoluble fraction of cultured mammalian cells, grown in the presence of 10 -4 M cold thymidine, is different from that of incorporation in the absence of cold thymidine. For quantitative estimation of net DNA synthesis in nonirradiated and irradiated cells, two methods were used: isolation of newly synthesized BUdR-labeled DNA by CsCl gradient centrifugation and a fluorometric estimation of DNA content in the synchronized population. Both methods showed that the depression of [ 3 H]thymidine incorporation in the presence of cold thymidine reflected a depression of net DNA synthesis. Radiosensitive steps in DNA synthesis were examined by the use of alkaline sucrose gradient centrifugation. The rate of replication along the DNA strands was inhibited to a lesser extent than that of over-all DNA synthesis. The labeling patterns of DNA exposed to [ 3 H]thymidine for 20 min indicated that ionizing radiation preferentially interfered with the formation of small-size 3 H-labeled DNA pieces. These results suggest that the initiation of DNA replication is more radiosensitive than the elongation of DNA strands whose replication has already been initiated. (U.S.)

  20. RBE comparison between rapid electrons of 20 MeV and 45 MeV with survival rate, DNA synthesis, DNA reparation and nucleoid sedimentation

    International Nuclear Information System (INIS)

    Alth, G.; Weniger, P.; Turtzer, K.; Klein, W.; Kocsis, F.; Krankenhaus der Stadt Wien-Lainz; Oesterreichisches Forschungszentrum Seibersdorf G.m.b.H. Inst. fuer Biologie)

    1982-01-01

    In order to find out possible differences of the biologic efficacy of rapid electrons of different energies, the authors examined the influence of rapid electrons of 20 MeV and 45 MeV upon the survival rate of Hela cells S3, their cell growth, DNA synthesis, DNA reparation, and sedimentation of nucleoids. The results show a difference only for the nucleoid sedimentation, i.e. there are more fractured DNA cords after 45 MeV irradiation. No significant differences could be demonstrated for the parameters of the survival curve, DNA synthesis and DNA reparation. Four double tests were carried out corresponding to the indicated types of examination. (orig.) [de

  1. Protein synthesis and sublethal damage repair in synchronized CHO cells

    International Nuclear Information System (INIS)

    Yezzi, M.J.; Tobias, C.A.; Blakely, E.A.

    1984-01-01

    The authors have previously reported that the split dose survival response to x-rays of asynchronous CHO-TSH1 cells is reduced if the cells are held at 40 0 C,a temperature that inhibits protein synthesis, for 2 hours before the first dose and during a 2-hour interval between doses. In conjunction with the survival experiments on asynchronous cells, the authors also examined the DNA rejoining ability in split dose studies with and without inhibition of protein synthesis. The results of these experiments suggest that inhibition of protein synthesis affects a pool of proteins that are necessary for the correct expression of the DNA, although they do not appear to be involved in rejoining DNA breaks. They have extended this work to the study of cells synchronized in G1 phase (2 hour post-mitosis) and S phase (10 hour post-mitosis). Autoradiographic analyses, using 3H-TdR pulse labeling, demonstrated that a delay in the progression of each synchronized cell population occurs after inhibition of protein synthesis. Data are reported on the effects of inhibition of protein synthesis on the ability of G1 and S phase cells to repair sublethal damage

  2. Elucidation of the mechanism of X-ray induced DNA duplication observed in human Gorlin cells

    International Nuclear Information System (INIS)

    Nomura, J.; Suzuki, N.; Kita, K.; Sugaya, S.

    2004-01-01

    A phenomenon in which DNA synthesis level increases rapidly after x-ray irradiation has found out in the cells which originate in Gorlin patients. A gene, by which an expression level changes after x-ray irradiation, is searched in the human Gorlin cells by the mRNA differential display method. The DNA synthesis level decreases in normal human cell after x-ray irradiation of 2 Gy dose, but increases twice in the Gorlin cell. Expression levels of gene SMT3A, however decrease clearly in the Gorlin cells after the irradiation. The relations between expression levels of gene SMT3M, a protein like ubichitin, and DNA synthesis levels are searched. DNA synthesis activity in normal human cells, which are treated by antisese oligonucleotide and suppressed expression of the genes SMT3A, increases after x-ray irradiation. An increase of the DNA synthesis level after the irradiation is not a phenomenon in particular cells, but indicates the possibility of general phenomena in normal human cells. It is reported that the gene SMT3A combines with a glycosylase which operates in DNA repairing process. The protein modification of gene SMT3A indicates a possibility for controlling of stress protection mechanism in the cells. (M. Suetake)

  3. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    Energy Technology Data Exchange (ETDEWEB)

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States); Rozengurt, Enrique, E-mail: erozengurt@mednet.ucla.edu [Division of Digestive Diseases, Department of Medicine, CURE: Digestive Diseases Research Center, David Geffen School of Medicine and Molecular Biology Institute, University of California at Los Angeles, CA (United States)

    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.

  4. DNA endoreduplication, RNA and protein synthesis during growth and development of the antheridial basal cell in Chara vulgaris L

    International Nuclear Information System (INIS)

    Malinowski, S.; Maszewski, J.

    1994-01-01

    Cytophotometric measurements of nuclear DNA contents and morphometric analyses indicate that the level of endo polyploidy plays an important role in determining the maximum size, transcriptional and translational activity that the antheridial basal cell attains during successive stages of spermatogenesis in Chara vulgaris. During the proliferative period of antheridial development, the metabolic activity of basal cell, expressed as the total incorporation of radioactive uridine and leucine was found to increase gradually with the increasing DNA C-values, yet both the synthesis of RNA and then the synthesis of proteins become reduced at the stage preceding spermiogenesis. In accordance with some earlier data, the obtained results seem to support the hypothesis that regulatory mechanisms of symplasmic connections between the antheridium and a thallus participate in the regulation of morphogenesis of the male sex organs in Chara. (author). 15 refs, 13 figs

  5. Laser-UV-microirradiation of Chinese hamster cells: the influence of the distribution of photolesions on unscheduled DNA synthesis

    International Nuclear Information System (INIS)

    Cremer, C.; Jabbur, G.

    1981-01-01

    Fibroblastoid Chinese hamster cells synchronized by mitotic selection were microirradiated in G1, using a low power laser-UV-microbeam (lambda = 257 nm). The incident energy was either concentrated on a small part of the nucleus (mode 1) or distributed over the whole nucleus (mode 11). Using the same incident UV energy, the local UV fluences were estimated to differ by two orders of magnitude. Following microirradiation the cells were incubated with [ 3 H]-thymidine for 2 h and thereafter processed for autoradiography. Silver grains were concentrated over the microirradiated part after mode 1 and distributed over the whole nucleus after mode 11 irradiation. To quantify the amount of unscheduled DNA synthesis, the number of grains per nucleus was determined. It increased with the total incident energy, but was not or only slightly affected by the mode of microirradiation, if appropriate autoradiographic conditions were used. The findings suggest that within the investigated range of energy densities (2.7-1000 J/m 2 ), the total amount of unscheduled DNA synthesis depends on the total number of pyrimidine dimers but not on their distribution in nuclear DNA. (author)

  6. Maintenance of DNA repair capacity in differentiating rat muscle cells in vitro

    International Nuclear Information System (INIS)

    Koval, T.M.; Kaufman, S.J.

    1981-01-01

    Unscheduled DNA synthesis was measured at several times during the differentiation of cultured rat skeletal muscle cells in response to exposures to 254 nm UV light. There was no change in the amount of repair DNA synthesis as the cells fuse and differentiate from postmitotic prefusion myoblasts to multinucleated contracting myotubes. (author)

  7. Effect of 5-fluorodeoxyuridine on DNA replication in ultraviolet-irradiated HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Brozmanova, J.; Masek, F.; Synzynys, B.I.; Saenko, A.S.

    1985-11-05

    In HeLa cells precultivated for 6 hours with 5-fluorodeoxyuridine (FUdR) and for 18 hours in FUdR-free medium, DNA synthesis was much more resistant to UV irradiation than that of untreated cells. DNA synthesized in FUdR-pretreated and UV irradiated cells represents a semiconservative DNA replication and shows more rapid shift of the pulse-labelled chased DNA to high molecular weight. This DNA synthesis is not induced by synchronization of the cell cycle. It is assumed that either the changes of chromatine structure, or an enhanced level of some enzymes might be involved in the replication of the damaged template. (author).

  8. Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury

    NARCIS (Netherlands)

    van Kleef, E. M.; Smits, J. F.; Schwartz, S. M.; Daemen, M. J.

    1996-01-01

    Infusion of angiotensin II (AngII) during the third and fourth week after balloon injury of the left common carotid artery of the rat induces smooth muscle cell (SMC) DNA synthesis. In this study we wanted to investigate whether alpha 1-adrenoreceptors are involved in AngII-induced SMC DNA synthesis

  9. DNA repair synthesis in rat retinal ganglion cells treated with chemical carcinogens or ultraviolet light in vitro, with special reference to aging and repair level

    International Nuclear Information System (INIS)

    Ishikawa, T.; Takayama, S.; Kitagawa, T.

    1978-01-01

    A system in which the retinal tissues of noninbred Wistar rats were used in combination with autoradiography was developed for measurement of DNA repair synthesis in ganglion cells of the central nervous system. Retinal tissues in short-term organ culture were treated with various carcinogens plus tritiated thymidine ([methyl -3 H]dThd) or were irradiated with uv light and then treated with [methyl -3 H]dThd. Preliminary study with retinal tissues from rats at various ages revealed no age-associated changes in the levels of unscheduled DNA synthesis in ganglion cells

  10. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

    Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

  11. H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

    Science.gov (United States)

    Stone, G. E.; Miller, O. L.; Prescott, D. M.

    1965-01-01

    The formation of a soluble H3-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H3-thymidine during G1 or G2. Uptake of H3-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H3-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H3-thymidine, the pool does not turn over during non-S periods. PMID:19866660

  12. On the effect of small radiation doses: Desoxyribonucleic acid (DNA) synthesis and DNA repair of thymus, spleen, and bone marrow cells in the rat after fractionated total body X-ray irradiation. Zur Wirkung kleiner Strahlendosen: Desoxyribonukleinsaeure-(DNA-)Synthese und DNA-Reparatur von Thymus-, Milz- und Knochenmarkszellen der Ratte nach fraktionierter Ganzkoerperroentgenbestrahlung

    Energy Technology Data Exchange (ETDEWEB)

    Tempel, K.; Ehling, G. (Muenchen Univ. (Germany, F.R.). Inst. fuer Pharmakologie, Toxikologie und Pharmazie)

    1989-09-01

    After three to seven days following to fractionated total body X-ray irradiation (TBI) (four expositions with doses of 0.3 to 5.0 cGy per fraction at intervals of 24 hours), a maximum 50 percent stimulation of the semiconservative DNA synthesis (SDS) of spleen cells was measured in vitro. This was not dependent of the fact if an acute high-dose (400 and/or 800 cGy) unique irradiation was applied after the fractionated TBI at the moment of stimulation. A significant increase of {sup 3}H-thymidine incorporation into the DNA of bone marrow and thymus cells was only found when doses of 1.25 cGy per fraction had been used. After fractionated TBI with doses of {ge}5 cGy per fraction, an increase of DNA synthesis resistant to hydroxyurea ('unprogrammed' DNA synthesis, UDS) was demonstrated in spleen cells. The UV-simulated UDS decreased proportionately. The sedimentation of thymus, spleen, and bone marrow nucleoids in a neutral saccharose gradient gave no evidence of an increased DNA repair capacity after fractionated TBI. Whereas the SDS stimulation by fractionated TBI with small doses can be explained by a modified proliferation behavior of exposed cells, the UDS behavior of spleen cells after considerably higher radiation doses suggests regenerative processes correlated with an increased number of cells resistant to hydroxyurea and cells presenting an UV repair deficiency. These findings can be considered to be a further proof of the assumed immune-stimulating effect of small radiation doses. (orig.).

  13. Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA

    International Nuclear Information System (INIS)

    Bodell, W.J.; Cleaver, J.E.; Roti Roti, J.L.

    1984-01-01

    The authors have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 0 C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 0 C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus, the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating steps(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 0 C with[ 3 H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 0 C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures. 23 references, 3 figures, 2 tables

  14. Psoralen plus ultraviolet radiation-induced inhibition of DNA synthesis and viability in human lymphoid cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Kraemer, K H; Waters, H L [National Cancer Inst., Bethesda, MD (USA); Ellingson, O L; Tarone, R E

    1979-08-01

    The present study investigated whether conditions of 8-methoxypsoralen (8-MOP) concentration and of exposure to high intensity long wavelength ultraviolet radiation (UV-A) during psoriasis and mycosis fungoides therapy might be sufficient to result directly in decreased lymphoid cell DNA synthesis and viability in vitro. Tritiated thymidine (/sup 3/HtdR) incorporation and cell growth following UV-A exposure alone or with 8-MOP was examined in peripheral blood lymphocytes and in Ebstein-Barr virus transformed human lymphoblastoid cell lines. UV-A exposure alone induced a dose-dependent inhibition of /sup 3/HTdR incorporation in both types of lymphoid cells. Pre-incubation with 0.1 ..mu..g/ml 8-MOP before UV-A exposure induced a significantly greater inhibition of /sup 3/HTdr incorporation. Further inhibition of /sup 3/HTdR incorporation was observed by preincubation of the lymphoblastoid cells with 1.0 ..mu..g/ml 8-MOP but not in the lymphocytes. The concentration of viable lymphoblastoid cells did not decrease below the original concentration after the highest dose of UV-A alone (29,00 J/m/sup 2/) but preincubation with 0.1 ..mu..g/ml 8-MOP resulted in 40% and 0.6% survival respectively after 3000 J/m/sup 2/. This study suggested that the low doses of 8-MOP and UV-A received by patients' lymphocytes may be sufficient to explain the decreased DNA synthesis found in their circulating leucocytes. (author).

  15. Reactivation of DNA replication of the parvovirus MVM in UV preirradiated mouse cells

    International Nuclear Information System (INIS)

    Vos, J.M.; Rommelaere, Jean

    1982-01-01

    The parvovirus Minute-Virus-of-Mice (MVM) was used to probe the DNA replication activities expressed by mouse fibroblasts. This system allowed us to study quantitatively the effect of UV-induced DNA lesions on the progression of DNA replication in vivo. MVM was UV-irradiated prior to infection. Pyrimidine dimers induced in the viral genome account for the reduced level of intracellular viral DNA synthesis, assuming that most of these lesions block viral DNA replication in unirradiated cells. The inhibition of damaged MVM DNA synthesis is less severe if the host cells themselves are irradiated prior to virus infection. This stimulation of viral DNA replication in pretreated cells might account for the UV-enhanced viral reactivation phenomenon, i.e. the increased survival of nuclear-replicating viruses propagated in cells preexposed to various genotoxic agents [fr

  16. Reactivation of DNA replication of the parvovirus MVM in UV preirradiated mouse cells

    Energy Technology Data Exchange (ETDEWEB)

    Vos, J.M.; Rommelaere, J. (Universite Libre de Bruxelles, Rhode-St-Genese (Belgium))

    1982-07-01

    The parvovirus Minute-Virus-of-Mice (MVM) was used to probe the DNA replication activities expressed by mouse fibroblasts. This system allowed us to study quantitatively the effect of UV-induced DNA lesions on the progression of DNA replication in vivo. MVM was UV-irradiated prior to infection. Pyrimidine dimers induced in the viral genome account for the reduced level of intracellular viral DNA synthesis, assuming that most of these lesions block viral DNA replication in unirradiated cells. The inhibition of damaged MVM DNA synthesis is less severe if the host cells themselves are irradiated prior to virus infection. This stimulation of viral DNA replication in pretreated cells might account for the UV-enhanced viral reactivation phenomenon, i.e. the increased survival of nuclear-replicating viruses propagated in cells preexposed to various genotoxic agents.

  17. DNA replication and repair in Tilapia cells. 1. The effect of ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yew, F.H.; Chang, L.M. (National Taiwan Univ., Taipei (China))

    1984-12-01

    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-..beta..-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor.

  18. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    International Nuclear Information System (INIS)

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells

  19. The effect of heat and radiation on the initiation and elongation processes of DNA synthesis

    International Nuclear Information System (INIS)

    Davies, R.C.; Bowden, G.T.; Cress, A.E.

    1983-01-01

    The pH step alkaline elution and alkaline sucrose gradient techniques were utilized to evaluate alterations in DNA replication (initiation and elongation) induced by heat and low dose X-irradiation in synchronized Chinese hamster ovary cells. The initiation and elongation processes of DNA synthesis were radioresistant at the G 1 /S boundary (4 hours after mitosis) while in mid S phase (9 hours after mitosis) DNA initiation and elongation were sensitive to X-irradiation. The initiation and elongation processes of DNA synthesis which were radiation resistant at the G 1 /S boundary could be inhibited by a hyperthermia treatment (43 0 C for 1 hour beginning at 4 hours after mitosis). The impairment of initiation in the heated cells was maintained through late S phase while that of elongation was reversible as judged by full recovery at 15 hours after mitosis. These data suggest that the known synergistic lethality of heat and radiation may be mediated by an impairment of initiation of DNA synthesis. (author)

  20. Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells

    Science.gov (United States)

    Lombardi, Maria; Castoria, Gabriella; Migliaccio, Antimo; Barone, Maria Vittoria; Di Stasio, Rosina; Ciociola, Alessandra; Bottero, Daniela; Yamaguchi, Hiroshi; Appella, Ettore; Auricchio, Ferdinando

    2008-01-01

    In breast cancer cells, cytoplasmic localization of the estradiol receptor α (ERα) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry. PMID:18644889

  1. Effects of the Nd:YAG laser on DNA synthesis and collagen production in human skin fibroblast cultures

    Energy Technology Data Exchange (ETDEWEB)

    Castro, D.J.; Abergel, R.P.; Meeker, C.; Dwyer, R.M.; Lesavoy, M.A.; Uitto, J.

    1983-09-01

    Human skin fibroblasts were subjected to treatment with a Neodymium:YAG laser at 1060 nm with varying levels of energy determined by a reproducible method of dosimetry. DNA synthesis in the cells was measured by the incorporation of (3H)thymidine, and collagen production was monitored by the synthesis of nondialyzable (3H)hydroxyproline after incubation of cells with (3H)proline. Using energy levels equal to 1.7 X 10(3) J/cm2, a significant reduction in DNA synthesis was noted, while the cells remained viable as tested by the trypan blue exclusion test. With energy levels higher or equal to 2.3 X 10(3) J/cm2, the suppression of DNA synthesis was accompanied by cell nonviability. The collagen production, when measured immediately following the treatment with 1.7 X 10(3) J/cm2, was markedly reduced, and similar effects were observed with higher energy levels. However, when the cells were tested for collagen production at 20 hours following laser treatment, there was a significant decrease in collagen production at energy levels as low as 1.1 X 10(3) J/cm2, a dose that did not affect DNA synthesis or cell viability. Thus, the results indicate that the Nd:YAG laser can selectively suppress collagen production without affecting cell proliferation. These observations suggest that laser treatment could potentially be used to reduce collagen deposition in conditions such as keloids and hypertrophic scars.

  2. Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

    NARCIS (Netherlands)

    A.J.R. de Jonge; W. Vermeulen (Wim); W. Keijzer; J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1985-01-01

    textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of

  3. Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

    NARCIS (Netherlands)

    M. Molinete; W. Vermeulen (Wim); A. Bürkle; J. Mé nissier-de Murcia; J.H. Küpper; J.H.J. Hoeijmakers (Jan); G. de Murcia

    1993-01-01

    textabstractThe zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during

  4. Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

    International Nuclear Information System (INIS)

    Hsia, M.T.

    1987-01-01

    The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs

  5. Influence of metronidazole on the survival rate of whole-body irradiated mice and on the DNA repair synthesis of lymphocytes

    International Nuclear Information System (INIS)

    Magdon, E.; Schroeder, E.

    1978-01-01

    With reference to literature reports the effect of Metronidazole [1-(hydroxyethyl)-5-nitro-2-methyl-imidazole] on the survival rate of C 3 H inbred mice following whole-body doses ranging from 5 to 15 Gy was determined under oxic and hypoxic conditions. Ehrlich ascites tumor cells were used to study the influence of Metronidazole on radiation-induced alterations of the DNA sedimentation behavior in the alkaline sucrose gradient under oxic conditions in vitro. The effect of Metronidazole on the semiconservative DNA synthesis was investigated under oxic and hypoxic conditions in Ehrlich ascites carcinoma cells and L5178Y lymphoma cells. Furthermore, it was examined whether the radiation-induced inhibition of semiconservative DNA synthesis in L5178Y lymphoma cells and the radiation-induced repair synthesis in lymphocytes is influenced by Metronidazole. From the values of the LDsub(50/30) after whole-body irradiation a sensitilization factor of 1.3 was derived for Metronidazole under hypoxic conditions. Under atmospheric conditions an increase of the radiation effect by a factor of 1.1 was obtained. The protective factor of hypoxia was 1.6 and thus greater than the radiosensibilization caused by Metronidazole. The DNA synthesis was slightly inhibited by Metronidazole under both hypoxic and euoxic conditions. The studies revealed no significant influence of Metronidazole on radiation-induced changes of the DNA sedimentation behavior and of the DNA repair synthesis as well as on the radiation induced inhibition of semiconservative DNA synthesis. (author)

  6. Control of DNA synthesis in inhibited and activated Agrostemma githago seeds

    Energy Technology Data Exchange (ETDEWEB)

    Hecker, M [Sektion Biologie, FG Algemeine Botanik und Pflanzenphysiologie, Universitaet Greifswald (German Democratic Republic)

    1975-01-01

    The relationships between DNA synthesis and germination capacity of Agrostemma seeds had been studied. Protein synthesis and RNA synthesis were activated at the very beginning of imbibition, whereas DNA synthesis started in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30 degC), or aged seeds with a low germination capacity were characterized by a significantly reduced protein synthesis. DNA synthesis was also reduced. The inhibition of the protein synthesis of Agrostemma embryos fed with cycloheximide or actinomycin D caused a depression of DNA synthesis. The results indicated that the initiation of DNA synthesis of imbibing Agrostemma seeds depended on the synthesis of special proteins. Abscisic acid inhibited the growth as well as DNA synthesis of isolated Agrostemma embryos. Nitomycin inhibited germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also showed a reduced incorporation of /sup 3/H-thymidine by DNA. It is suggested that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, was involved in the mechanism of ripening of the Agrostemma seeds.

  7. Control of DNA synthesis in inhibited and activated Agrostemma githago seeds

    International Nuclear Information System (INIS)

    Hecker, M.

    1975-01-01

    The relationships between DNA synthesis and germination capacity of Agrostemma seeds had been studied. Protein synthesis and RNA synthesis were activated at the very beginning of imbibition, whereas DNA synthesis started in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30 degC), or aged seeds with a low germination capacity were characterized by a significantly reduced protein synthesis. DNA synthesis was also reduced. The inhibition of the protein synthesis of Agrostemma embryos fed with cycloheximide or actinomycin D caused a depression of DNA synthesis. The results indicated that the initiation of DNA synthesis of imbibing Agrostemma seeds depended on the synthesis of special proteins. Abscisic acid inhibited the growth as well as DNA synthesis of isolated Agrostemma embryos. Nitomycin inhibited germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also showed a reduced incorporation of 3 H-thymidine by DNA. It is suggested that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, was involved in the mechanism of ripening of the Agrostemma seeds. (author)

  8. The effect of vinblastine on DNA metabolism in tumour cells

    International Nuclear Information System (INIS)

    Cabela, E.; Klein, W.

    1976-01-01

    Studies on the influence of Vinblastine on normal and tumor cells show that semiconservative DNA-synthesis correlates with the enzymatic activity of thymidinkinase. DNA-repair-investigations performed with Yoshida-Ascites cells indicate an inhibition effect on the ligase system after Vinblastine-treatment and gamma irradiation. (author)

  9. Measurement of the capability of DNA synthesis of human fetal liver cells by the assay of 3H-TdR incorporation

    International Nuclear Information System (INIS)

    Wang Tao; Ma Xiangrui; Wang Hongyun; Cao Xia

    1987-01-01

    The fetal liver is one of the major sites of hematopoiesis during gestation. Under erythropoietin (EPO) stimulation, in erythroid precusor cells of fetal liver, proliferation and differentiation occurred and function of metabolism was enhanced. The technique of 3 H-TdR incorporation was used to measure the function of fetal liver cellular DNA synthesis. As EPO concentration at the range of approximately 20 ∼ 100 mU/ml, the counts of 3 H-TdR incorporation into fetal liver cells increased. As the concentration of EPO increased, however, its incorporation counts are lower than that in bone marrow of either the fetal or the adult. It suggested that precusors of erythrocyte of fetal liver has differentiated to later phases with the progressive accumulation of mature cells, therefore, both proliferation and function of metabolism are more or less decreased respectively. Under EPO stimulation, however, precusor of erythroid of fetal liver can greatly increase potential effects on DNA synthesis

  10. In vitro test systems for the identification of gentoxic chemicals in the human environment: The proof of DNA repair synthesis in liver cells

    International Nuclear Information System (INIS)

    Rossberger, S.

    1986-01-01

    This work examines the possibilities of proving a DNA repair by gentoxic chemicals in primary hepatozytes and 2sFou liver cells of rates. Two different processes used for the in vitro mutagenic testing of alien substances for determining the DNA repair synthesis in primary hepatozytes, in the autoradiographic method and the gradient centrifuging method, are compared regarding their reliability and sensitivity. The rat hepatom cell line 2sFou was examined for its suitability for proving chemically induced DNA repair, instead of primary hepatozytes. (orig./MG) [de

  11. Opportunities for measuring DNA synthesis time by quantitative autoradiography

    International Nuclear Information System (INIS)

    Vasileva, D.

    1980-01-01

    DNA sysntesis time (Tsub(s)) in cells of the canine erythropoiesis and myelopoiesis pools was determined by quantitative autoradiography according to Doermer. In contrast to mitosis labelling for Tsub(s) estimation as so far applied, this technique uses well-differentiated cells. After blocking endogeneous DNA synthesis with 5-fluorodeoxyuridine, its further course becomes dependent on exogeneous supply of thymidine, in the form of 14 C-thymidine. From incroporation of the latter into the individual cell within a definite time span (3-7 min) and taking into account its total amount, Tsub(s) may be calculated. The data thus obtained were found to agree with Tsub(s) values as estimated from the labelled mitosis curve

  12. Unscheduled DNA synthesis after β-irradiation of mouse skin in situ

    International Nuclear Information System (INIS)

    Ootsuyama, Akira; Tanooka, Hiroshi

    1986-01-01

    The skin of ICR mouse was irradiated with β-rays from 90 Sr- 90 Y with surface doses up to 30 krad. Unscheduled DNA synthesis (UDS) was measured by autoradiography after labeling the skin with radioactive thymidine using the forceps-clamping method. The level of UDS in epithelial cells of the skin was detected as an increasing function of radiation dose. Fibroblastic cells, compared with epithelial cells and hair follicle cells at the same depth of the skin, showed a lower level of UDS, indicating a lower DNA repair activity in fibroblasts. Cancer risk of the skin was discussed. (Auth.)

  13. Inhibition of polymerases-alpha and -beta completely blocks DNA repair induced by UV irradiation in cultured mouse neuronal cells

    International Nuclear Information System (INIS)

    Licastro, F.; Sarafian, T.; Verity, A.M.; Walford, R.L.

    1985-01-01

    The effects of hydroxyurea, aphidicolin and dideoxythymidine on UV-induced DNA repair of mouse neuronal granular cells were studied. Aphidicolin, which is considered a specific inhibitor of polymerase-alpha, decreased spontaneous DNA synthesis by 93% and totally suppressed DNA repair. Dideoxythymidine, an inhibitor of polymerase-beta, was more potent in decreasing scheduled DNA synthesis than aphidicolin, and also completely blocked the UV-induced DNA repair. Hydroxyurea, a specific inhibitor of ribonucleotide reductase, inhibited scheduled DNA synthesis, but unscheduled DNA synthesis after UV irradiation was always well detectable. Our data suggest that in neuronal cells from 5 to 10 days old mice both polymerases-alpha and -beta are required for both DNA synthesis and repair. These two enzymes may act jointly in filling up the gaps along the DNA molecule and elongating the DNA chain

  14. Novel pattern of post-γ ray de novo DNA synthesis in a radioresistant human strain

    International Nuclear Information System (INIS)

    Mirzayans, R.; Gentner, N.E.; Paterson, M.C.

    1985-01-01

    Enhanced resistance to radiation cytotoxicity in a fibroblast strain from an afflicted member of a Li-Fraumeni syndrome family may be largely ascribable to a change in the pattern of DNA replicative synthesis following γ ray exposure. That is, the extent of the initial radiogenic inhibition of replicative synthesis and the time interval before its subsequent recovery were both found to be greater in radioresistant (RR) compared to normal cells. In addition, the post-recovery replication rates in the RR cells were both higher and longer lasting than those in the control cells. A similar differential pattern was also seen following treatment with 4NQO, another DNA-damaging agent to which this RR strain displays enhanced resistance. Moreover, several conventional DNA repair assays indicated that the RR cells repair radiogenic damage at normal rates. The authors therefore suggest that the increased inhibition and prolonged lag in resumption of replicative synthesis seen in the RR strain upon exposure to certain genotoxic agents may enhance cellular recovery by ''buying additional time'' for processing of potentially lethal lesions

  15. DnaB gene product-independence of DNA polymerase III-directed repair synthesis in Escherichia coli K-12

    International Nuclear Information System (INIS)

    Billen, D.; Hellermann, G.R.

    1977-01-01

    An investigation has been carried out into the role of dnaB gene product in X-ray-induced repair synthesis carried out by DNA polymerase III in toluene-treated Escherichia coli K-12. A polAl polBlOO dnaB mutant deficient in both DNA polymerase I and II activities was used, and it was shown that the level of X-ray-induced, ATP-dependent, non-conservative DNA synthesis was, unlike semi-conservative DNA synthesis, unaffected by a temperature shift from 30 0 to 42 0 C. The dnaB gene product was not therefore necessary for DNA polymerase III-directed repair synthesis, which occurred in the absence of replicative synthesis. (U.K.)

  16. Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

    Science.gov (United States)

    Murray, Heath; Koh, Alan

    2014-10-01

    In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

  17. Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells

    International Nuclear Information System (INIS)

    Hodge, B.O.; Kream, B.E.

    1988-01-01

    We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of [ 3 H]proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normal bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis

  18. Effects of lithium chloride as a potential radioprotective agent on radiation response of DNA synthesis in mouse germinal cells.

    Science.gov (United States)

    Bhattacharjee, D; Rajan, R; Krishnamoorthy, L; Singh, B B

    1997-06-01

    Mouse spermatogonial germ cells are highly sensitive to ionizing radiation. Lithium salts are reported to stimulate the postirradiation recovery of hematopoietic marrow cells. We have, therefore, examined whether administered lithium chloride (LiCl) would also be able to protect the mouse germinal cells against radiation injury. Taking DNA synthesis as an endpoint, our results show that the testicular DNA-specific activity in irradiated mice was higher by 61% on average when they had been pretreated with LiCl both 24 h and 1 h prior to gamma-irradiation (2.0 Gy). It was also observed that the DNA synthetic activity in the germinal cells fully recovered after LiCl pretreatment at doses of 40 mg per kg body weight prior to total body irradiation of 0.05-0.25 Gy, whereas at doses of 0.5-6.0 Gy, following the same procedure of LiCl pretreatment, only an incomplete recovery was observed. The dose reduction factor for LiCl is 1.84. The current findings indicate that pretreatment with LiCl provides considerable protection against radiation damage in mouse spermatogonia.

  19. A UV-resistant mutant without an increased repair synthesis activity, established from a UV-sensitive human clonal cell line

    International Nuclear Information System (INIS)

    Suzuki, N.

    1984-01-01

    When cells of a human clonal cell line, RSa, with high sensitivity to UV lethality, were treated with the mutagen, ethyl methanesulfonate, a variant cell strain, UVr-1, was established as a mutant resistant to 254-nm far-ultraviolet radiation (UV). Cell proliferation studies showed that UVr-1 cells survived and actively proliferated at doses of UV-irradiation that greatly suppressed the proliferation of RSa cells. Colony-formation assays also confirmed the increased resistance of UVr-1 cells to UV. The recovery from a UV-induced inhibition in DNA synthesis, as [methyl- 3 H]thymidine uptake into cellular DNA, was more pronounced in UVr-1 cells than in RSa cells. Nevertheless, there was no significant difference in the activity of UV-induced DNA repair synthesis in either cell line, as estimated by the extent of unscheduled DNA synthesis and DNA repair replication. These characteristics of UVr-1 cells are discussed in the light of a previously reported UV-resistant variant, UVr-10, which had an increased DNA repair synthesis activity. (Auth.)

  20. Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate

    International Nuclear Information System (INIS)

    Williams, J.I.; Friedberg, E.C.

    1982-01-01

    Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m 2 . The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine. (author)

  1. Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

    Science.gov (United States)

    Fillingame, R H; Jorstad, C M; Morris, D R

    1975-01-01

    There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication. PMID:1060087

  2. DNA repair in lens cells during chick embryo development

    International Nuclear Information System (INIS)

    Counis, M.F.; Chaudun, E.; Simonneau, L.; Courtois, Y.

    1979-01-01

    When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenon of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [ 3 H)thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in the experimental system the synthesis of delta- and αcrystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case. (Auth.)

  3. Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

    International Nuclear Information System (INIS)

    Zurlo, J.; Mignano, J.E.; Eustice, D.C.; Poirier, M.C.; Yager, J.D.

    1986-01-01

    One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. It was found that N-OH-AAF caused a dose-dependent inhibition of [ 3 H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF were cultured. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. The results show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with previous studies support the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis. (Auth.)

  4. [Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA].

    Science.gov (United States)

    Jia, Xiao-Wei; Zhang, Guo-Hui; Shi, Hai-Yan

    2012-12-01

    Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. kod-ssb may in future be used to enhance DNA and cDNA amplification.

  5. Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

    International Nuclear Information System (INIS)

    Mullenders, L.H.F.

    1983-01-01

    The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after iiradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S 1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop. (Auth.)

  6. Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

    Energy Technology Data Exchange (ETDEWEB)

    Mullenders, L.H.F. (Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica en Chemische Mutagenese); Zeeland, A.A. van; Natarajan, A.T. (Cohen (J.A.) Inst. voor Radiopathologie en Stralenbescherming, Leiden (Netherlands))

    1983-09-09

    The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S/sub 1/ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.

  7. [Effect of Mn(II) on the error-prone DNA polymerase iota activity in extracts from human normal and tumor cells].

    Science.gov (United States)

    Lakhin, A V; Efremova, A S; Makarova, I V; Grishina, E E; Shram, S I; Tarantul, V Z; Gening, L V

    2013-01-01

    The DNA polymerase iota (Pol iota), which has some peculiar features and is characterized by an extremely error-prone DNA synthesis, belongs to the group of enzymes preferentially activated by Mn2+ instead of Mg2+. In this work, the effect of Mn2+ on DNA synthesis in cell extracts from a) normal human and murine tissues, b) human tumor (uveal melanoma), and c) cultured human tumor cell lines SKOV-3 and HL-60 was tested. Each group displayed characteristic features of Mn-dependent DNA synthesis. The changes in the Mn-dependent DNA synthesis caused by malignant transformation of normal tissues are described. It was also shown that the error-prone DNA synthesis catalyzed by Pol iota in extracts of all cell types was efficiently suppressed by an RNA aptamer (IKL5) against Pol iota obtained in our work earlier. The obtained results suggest that IKL5 might be used to suppress the enhanced activity of Pol iota in tumor cells.

  8. Sister chromatid exchanges in X-ray irradiated blood lymphocytes from patients with hereditary diseases with radioresistant DNA synthesis

    International Nuclear Information System (INIS)

    Pleskach, N.M.; Andriadze, M.I.; Mikhel'son, V.M.; Zhestyanikov, V.D.

    1988-01-01

    X-ray irradiation induced sister chromatid exchanges (SCE) in blood lymphocytes from patient with Down's syndrome and adult progeria (in both the cases radioresistant DNA synthesis takes place). In normal lymphocytes (in which ionizing radiation inhibits the replicative synthesis of DNA) the rate of SCE rises with the rise of radiation dose. Thus, the rate of SCE in X-ray irradiated lymphocytes is in reverse dependence with radioresistance of replicative synthesis of DNA. The data obtained are explained in accordance with the replicative hypothesis of the SCE nature (Painter, 1980a): in cells of patients with Down's syndrome, xeroderma pigmentosum from 2 and progeria of adults the time of existence of partly replicated clusters of replicons is decreased due to radioresistant replicative synthesis of DNA, but the presence of partly replicated clusters of replicons in necessary for SCE formation. Therefore the rate of SCF in X-irradiated cells of these patients decreases

  9. On the recovery of the DNA-synthesis after X-irradiation in the spleen of mice and its modification by the NAD-metabolism

    International Nuclear Information System (INIS)

    Streffer, C.

    1974-01-01

    The incorporation of tritium-labelled thymidine into the DNA of mice spleen cells after whole body irradiation with X-rays was measured in order to study the decrease of DNA synthesis is decreased for several hours after irradiation with low doses. Recovery effects become operative after six hours. The radiation effect on the NAD metabolism, known to be related to DNA synthesis, was also investigated. The rate of NAD synthesis is influenced via the extremely radiosensitive metabolic process in the nucleus. Conversely, inhibition of DNA synthesis by injection of NAD enhances the recovery of DNA synthesis after irradiaton. (G.G.)

  10. Effects of low doses of gamma radiation on DNA synthesis in the developing rat brain

    International Nuclear Information System (INIS)

    Cerda, H.

    1983-01-01

    Rats of one or ten days of age were irradiated with low doses of gamma radiation, and synthesis of DNA was examined by the incorporation of 3 H-thymidine in the cerebellum and the rest of the brain in vivo. DNA synthesis was depressed in both parts of the brain but the effects were larger in cerebellum. A minimum was found about 10 hours after irradiation in the older rats and later (18 h) in the younger ones. The dose response in 10 day-old rats, was biphasic and showed that cerebellum was more affected. Autoradiographs showed that fewer cells entered the cycle and those synthesizing showed a depressed rate of synthesis. These findings are discussed in relation to induction of cell death. (Auth.)

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Multiple regulatory systems coordinate DNA replication with cell growth in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Heath Murray

    2014-10-01

    Full Text Available In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.

  13. Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

    Science.gov (United States)

    Murray, Heath; Koh, Alan

    2014-01-01

    In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. PMID:25340815

  14. Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

    International Nuclear Information System (INIS)

    Burkovics, Peter; Sebesta, Marek; Kolesar, Peter; Sisakova, Alexandra; Marini, Victoria; Plault, Nicolas; Szukacsov, Valeria; Pinter, Lajos; Haracska, Lajos; Robert, Thomas; Kolesar, Peter; Gangloff, Serge; Krejci, Lumir

    2013-01-01

    Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. (authors)

  15. DNA repair studies in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.; Owens, J.G.

    1984-01-01

    In submammalian test systems, nitrosocarbamates (NEC) are 100-fold more mutagenic than are their corresponding nitrosourea homologues. To learn more about its interaction with germ-cell DNA in the mouse testis, male mice were given i.p. injections of NEC. Testicular injections of [ 3 H]dThd were given along with the NEC. Sixteen days after treatment, sperm were recovered from the caudal epididymides and assayed for an unscheduled-DNA-synthesis

  16. Unscheduled DNA synthesis in human skin after in vitro ultraviolet-excimer laser ablation

    International Nuclear Information System (INIS)

    Green, H.A.; Margolis, R.; Boll, J.; Kochevar, I.E.; Parrish, J.A.; Oseroff, A.R.

    1987-01-01

    DNA damage repaired by the excision repair system and measured as unscheduled DNA synthesis (UDS) was assessed in freshly excised human skin after 193 and 248 nm ultraviolet (UV)-excimer laser ablative incisions. Laser irradiation at 248 nm induced DNA damage throughout a zone of cells surrounding the ablated and heat-damaged area. In contrast, with 193 nm irradiation UDS was not detected in cells adjacent to the ablated area, even though DNA strongly absorbs this wavelength. Our results suggest that the lack of UDS after 193 nm irradiation is due to: ''shielding'' of DNA by the cellular interstitium, membrane, and cytoplasm, DNA damage that is not repaired by excision repair, or thermal effects that either temporarily or permanently inhibit the excision repair processes

  17. DNA labeled during phosphonoacetate inhibition and following its reversal in herpesvirus infected cells

    International Nuclear Information System (INIS)

    Jacob, R.J.

    1984-01-01

    Human embryonic lung cells were pre-equilibrated with phosphonoacetate and 32 P orthophosphate label, then infected with phosphonoacetate-sensitive herpes simplex virus (HSV) type 1. Analyses of viral DNA produced in these cells showed the following. i) Viral DNA was synthesized in infected cells exposed to 100 μg of the drug per ml of medium but not in cells exposed to four-fold higher concentrations of the drug. ii) At 300 μg/ml a region of the DNA between 0.58 and 0.69 map units became transiently labeled, but the restriction endonuclease fragment containing these sequences migrated more slowly than the corresponding fragment from virion DNA. iii) Viral DNA extracted from infected cells 1.5 hours post drug withdrawal (300 μg/ml) was preferentially labeled in 2 regions of the genome mapping between 0.17 and 0.23 and 0.58-0.69 map units. This finding is in agreement with a report of Friedman et al. suggesting that HSV DNA contains two different sites if initiation. In addition a 4.8 x 10 6 molecular weight fragment was also preferentially labeled. This fragment could represent a smaller, aberrantly migrating fragment from the 0.17-0.27 map unit region of the DNA. iv) Viral DNA extracted from infected cells at longer intervals after drug withdrawal showed an increasing gradient of radioactivity progressively labeling the genome. These results are consistent with the hypothesis that viral DNA has at least two sites of initiation of DNA synthesis and that both sites are within the L component of the DNA. Alternatively, the results could be interpreted as two sites of localized synthesis (repair) that are detected at high concentrations of phosphonoacetate and immediately following reversal of inhibition of DNA synthesis. The results do not exclude the possibility that secondary sites in both L and S are utilized late in infection or in untreated cells. (Author)

  18. DNA repair synthesis dependent on the uvrA,B gene products

    International Nuclear Information System (INIS)

    Moses, R.E.; Moody, E.E.M.

    1975-01-01

    Ultraviolet irradiation of toluene-treated Escherichia coli causes an inhibition of replicative DNA synthesis. This is followed by the appearance of nonconservative DNA repair synthesis which does not require either the polymerase or 5' → 3' exonucleolytic activities of DNA polymerase I. The repair synthesis may be catalyzed by DNA polymerase III activity but does not require a functional DNA polymerase II. The ultraviolet-induced synthesis requires ATP and is dependent on a functional uvrA and uvrB gene product. However, other uvr gene products are not required for the synthesis. The recB function is also not required

  19. UV-induced DNA-binding proteins in human cells

    International Nuclear Information System (INIS)

    Glazer, P.M.; Greggio, N.A.; Metherall, J.E.; Summers, W.C.

    1989-01-01

    To investigate the response of human cells to DNA-damaging agents such as UV irradiation, the authors examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m 2 , the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification

  20. Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

    Energy Technology Data Exchange (ETDEWEB)

    Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

    1997-03-01

    Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

  1. Effects of lithium chloride as a potential radioprotective agent on radiation response of DNA synthesis in mouse germinal cells

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, D. [Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Rajan, R. [Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Krishnamoorthy, L. [Kidwai Memorial Institute of Oncology, Bangalore 560 029 (India); Singh, B.B. [Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)

    1997-06-01

    Mouse spermatogonial germ cells are highly sensitive to ionizing radiation. Lithium salts are reported to stimulate the postirradiation recovery of hematopoietic marrow cells. We have, therefore, examined whether administered lithium chloride (LiCl) would also be able to protect the mouse germinal cells against radiation injury. Taking DNA synthesis as an endpoint, our results show that the testicular DNA-specific activity in irradiated mice was higher by 61% on average when they had been pretreated with LiCl both 24 h and 1 h prior to {gamma}-irradiation (2.0 Gy). It was also observed that the DNA synthetic activity in the germinal cells fully recovered after LiCl pretreatment at doses of 40 mg per kg body weight prior to total body irradiation of 0.05-0.25 Gy, whereas at doses of 0.5-6.0 Gy, following the same procedure of LiCl pretreatment, only an incomplete recovery was observed. The dose reduction factor for LiCl is 1.84. The current findings indicate that pretreatment with LiCl provides considerable protection against radiation damage in mouse spermatogonia. (orig.). With 3 tabs.

  2. DNA nanotechnology from the test tube to the cell.

    Science.gov (United States)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology - applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems - lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  3. DNA nanotechnology from the test tube to the cell

    Science.gov (United States)

    Chen, Yuan-Jyue; Groves, Benjamin; Muscat, Richard A.; Seelig, Georg

    2015-09-01

    The programmability of Watson-Crick base pairing, combined with a decrease in the cost of synthesis, has made DNA a widely used material for the assembly of molecular structures and dynamic molecular devices. Working in cell-free settings, researchers in DNA nanotechnology have been able to scale up system complexity and quantitatively characterize reaction mechanisms to an extent that is infeasible for engineered gene circuits or other cell-based technologies. However, the most intriguing applications of DNA nanotechnology -- applications that best take advantage of the small size, biocompatibility and programmability of DNA-based systems -- lie at the interface with biology. Here, we review recent progress in the transition of DNA nanotechnology from the test tube to the cell. We highlight key successes in the development of DNA-based imaging probes, prototypes of smart therapeutics and drug delivery systems, and explore the future challenges and opportunities for cellular DNA nanotechnology.

  4. Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Ming Ming

    Full Text Available 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.

  5. Radiation sensitizations at DNA-level by chemical and biological agents. Coordinated programme on improvement of radiotherapy of cancer using modifiers of radiosensitivity of cells

    International Nuclear Information System (INIS)

    Altmann, H.

    1982-01-01

    Radiation sensitization by chemical agents at DNA level is discussed. Procaine, Halothan and Metronidazole showed no significant effect on unscheduled DNA synthesis (UDS) in mouse spleen cells, investigated by autoradiography and no effect on rejoining of DNA single strand breaks after gamma or UV irradiation. Oxyphenbutazon and prednisolone reduced the replicative DNA synthesis in vitro and in vivo but there was only little effect on DNA repair in the in vivo experiments. These two substances showed also a small reduction in poly(ADP-ribose) synthesis (PAR synthesis). 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in combination with UV irradiation showed that 5-MOP was more toxic than mutagen, but induced much less DNA crosslinks than 8-MOP. Autoradiographic studies of radiation sensitization by biological agents showed significant inhibition of UDS in Yoshida tumor cells after acute mycoplasma infection in rats. Nucleoid sedimentation studies showed only in the case of Yoshida tumor cells after mycoplasma infection a dramatic effect in the sedimentation behaviour. Sensitization of cells by changing chromatin structure was also studied. Benzamide, 3-NH 2 -benzamide, 3-Methoxybenzamide, Spermine, Theophyllin and Caffeine were tested in different concentrations on replicative DNA synthesis, UDS after UV irradiation and PAR synthesis Chinese hamster ovary cells. 5-Methoxybenzamide was the strongest sensitizer and inhibitor of the PAR synthesis, and was used in further experiments. Results of KFA Juelich on sensitization of a mamma-adenocarcinoma EO 771 on C57 B1 mice are given. Replicative DNA synthesis, DNA repair and PAR synthesis were compared in spleen cells and adenocarcinoma cells after treatment with 5-Methoxybenzamide. An inhibitory effect on UDS could be shown only in adenocarcinoma cells but not in the mice spleen cells

  6. Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA

    International Nuclear Information System (INIS)

    Kastan, M.B.; Gowans, B.J.; Lieberman, M.W.

    1982-01-01

    Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6- 3H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approximately 2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of approximately 2.7% in 10-20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites

  7. Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

    Science.gov (United States)

    Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

    2018-01-01

    DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

  8. Stimulation of DNA synthesis in bacterial DNA-membrane complexes after low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Watkins, D K [Hammersmith Hospital, London (UK). M.R.C. Experimental Radiopathology Unit

    1980-09-01

    DNA-membrane complexes from three strains of E. coli were irradiated and changes in the rates of DNA synthesis were observed. Doses from 1-10 krad to complexes from W3110 and pol A1 strains gave up to a 100 per cent increase in DNA synthesis; under the same conditions, no change was observed in Bsub(s-1). The degree of stimulation did not depend on the presence of oxygen during irradiation, and a post-irradiation incubation was necessary to achieve activation. The properties of all three complexes were similar when unirradiated. Irradiation of intact organisms under conditions which produced marked, oxygen-dependent inhibition of the Bsub(s-1) complex had no significant effect on those from W3110 and pol A1. Enhanced DNA synthesis is concluded to be due wholly to repair of pre-existing DNA. It is further postulated that DNA synthesis in untreated complexes (E.coli B's,W3110 and pol A1) is mainly of the repair-type and does not necessarily take place at the site of DNA-membrane attachment.

  9. Distribution of ultraviolet-induced DNA repair synthesis in nuclease sensitive and resistant regions of human chromatin

    International Nuclear Information System (INIS)

    Smerdon, M.J.; Tlsty, T.D.; Lieberman, M.W.

    1978-01-01

    The distribution of ultraviolet radiation (uv) induced DNA repair synthesis within chromatin was examined in cultured human diploid fibroblasts (IMR-90). Measurement of the time course of repair synthesis yielded two distinct phases: An initial rapid phase (fast repair) which occurs during the first 2 to 3 h after damage and a slower phase (slow repair) associated with a tenfold decrease in the rate of nucleotide incorporation, which persists for at least 35 h after damage. Staphylococcal nuclease digests of nuclei from cells damaged with uv and labeled during the fast-repair phase revealed a marked preference of fast-repair synthesis for the nuclease-sensitive regions. A new method was developed to analyze the digestion data and showed that approximately 50% of the nucleotides incorporated during the fast-repair phase are located in staphylococcal nuclease-sensitive regions, which comprise about 30% of the genome. Calculations from these data indicate that in the staphylococcal nuclease-sensitive regions the number of newly inserted nucleotides per unit DNA is about twice that of resistant regions. These results were supported by electrophoresis studies which demonstrated a decreased representation of fast-repair synthesis in core particle DNA. In contrast, the distribution within chromatin of nucleotides incorporated during the slow-repair phase was found to be much more homogeneous with about 30% of the repair sites located in 25% of the genome. Digestion studieswith DNase I indicated a slight preference of repair synthesis for regions sensitive to this enzyme; however, no marked difference between the distributions of fast- and slow-repair synthesis was observed. This study provides evidence that the structural constraints placed upon DNA in chromatin also place constraints upon uv-induced DNA repair synthesis in human cells

  10. Unscheduled DNA synthesis in human skin after in vitro ultraviolet-excimer laser ablation

    Energy Technology Data Exchange (ETDEWEB)

    Green, H.A.; Margolis, R.; Boll, J.; Kochevar, I.E.; Parrish, J.A.; Oseroff, A.R.

    1987-08-01

    DNA damage repaired by the excision repair system and measured as unscheduled DNA synthesis (UDS) was assessed in freshly excised human skin after 193 and 248 nm ultraviolet (UV)-excimer laser ablative incisions. Laser irradiation at 248 nm induced DNA damage throughout a zone of cells surrounding the ablated and heat-damaged area. In contrast, with 193 nm irradiation UDS was not detected in cells adjacent to the ablated area, even though DNA strongly absorbs this wavelength. Our results suggest that the lack of UDS after 193 nm irradiation is due to: ''shielding'' of DNA by the cellular interstitium, membrane, and cytoplasm, DNA damage that is not repaired by excision repair, or thermal effects that either temporarily or permanently inhibit the excision repair processes.

  11. DNA synthesis in toluene-treated bacteriophage-infected minicells of Bacillus subtilis

    International Nuclear Information System (INIS)

    Amann, E.; Reeve, J.N.

    1978-01-01

    Bateriophage (phi29, SPP1, or SP01)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [ 3 H]dTTP into a trichloroacetic acid-precipitable form. The [ 3 H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2.10 5 ). Short exposure of the DNA molecules containing the incorporated [ 3 H]dTMP to Escherichia coli exonuclease III results in over 90% of the [ 3 H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil. (Auth.)

  12. Restoration of ultraviolet-induced unscheduled DNA synthesis of xeroderma pigmentosum cells by the concomitant treatment with bacteriophage T4 endonuclease V and HVJ (Sendai virus)

    International Nuclear Information System (INIS)

    Tanaka, K.; Sekiguchi, M.; Okada, Y.

    1975-01-01

    Ultraviolet (uv)-induced unscheduled DNA synthesis of xeroderma pigmentosum cells, belonging to complementation groups, A, B, C, D, and E, was restored to the normal level by concomitant treatment of the cells with T4 endonuclease V and uv-inactivated HVJ (Sendai virus). The present results suggest that T4 endonuclease molecules were inserted effectively into the cells by the interaction of HVJ with the cell membranes, the enzyme was functional on human chromosomal DNA which had been damaged by uv irradiation in the viable cells, all the studied groups of xeroderma pigmentosum (variant was not tested) were defective in the first step (incision) of excision repair

  13. Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

    International Nuclear Information System (INIS)

    Dai Heqiao; Liu Jianying; Malkas, Linda H.; Catalano, Jennifer; Alagharu, Srilakshmi; Hickey, Robert J.

    2009-01-01

    Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC 50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC 50 = 88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication

  14. The relationship between DNA synthesis and incorporation of (14C) lysine into different histone fractions in Ehrlich ascites tumour cells

    International Nuclear Information System (INIS)

    Malec, J.; Kornacka, L.; Wojnarowska, M.; Moscicka, M.

    1974-01-01

    The effect of inhibition of DNA synthesis by hydroxyurea on ( 14 C) lysine incorporation into the main four histone fractions in Ehrlich ascites tumor cells, was examined in vitro. The radioactivity of lysine-rich histones, especially of histone f1, was preferentially decreased. The smallest decrease was observed for histone f3. The incorporation into other cellular proteins was but slightly affected. (author)

  15. Scintillometric determination of DNA repair in human cell lines. A critical appraisal

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, V.; Zantedeschi, A.; Levis, A.G. (Padua Univ. (Italy). Ist. di Biologica Animale); Nuzzo, F.; Stefanini, M. (Consiglio Nazionale delle Ricerche, Pavia (Italy). Ist. di Genetica Biochimica ed Evoluzionistica); Abbondandolo, A.; Bonatti, S.; Fiorio, R.; Mazzaccaro, A. (Consiglio Nazionale delle Ricerche, Pisa (Italy). Ist. di Mutagenesi e Differenziamento); Capelli, E. (Pavia Univ. (Italy). Ist. di Genetica)

    1982-04-01

    The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with (/sup 3/H)thymidine, the radioactivity incorporated into DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (Csub(HU), Tsub(HU)). The ratios Tsub(HU)/Csub(HU) and Tsub(HU)/T:Csub(HU)/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation.

  16. Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Konze-Thomas, B.; Hazard, R.M.; Maher, V.M.; McCormick, J.J. (Michigan State Univ., East Lansing (USA). Carcinogenesis Lab.)

    1982-01-01

    Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G/sub 0/ state. Autoradiography studies of cells released from G/sub 0/ after 72 h and replated at lower densities (3-9 x 10/sup 3/ cells/cm/sup 2/) in fresh medium showed that semiconservative DNA synthesis (S phase) began approx. equal to 24 h after the replating. The task was to determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts.

  17. Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Ockey, C.H.

    1982-04-01

    DNA fibre autoradiography, after incorporation of high specific activity /sup 3/H-thymidine and /sup 3/H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) of G/sub 1/ (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G/sub 1/ cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.

  18. Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec- mutant of Escherichia coli K12

    International Nuclear Information System (INIS)

    Pirsel, M.; Slezarikova, V.

    1977-01-01

    A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA - ) or by chloramphenicol (CAP) treatment prior to UV irradiation (2.5 J m -2 ) increased more than tenfold the resistance of the strain Escherichia coli K12 SR19 to UV radiation. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation, and a higher stability of both parental and newly synthesized DNAs could be demonstrated. (author)

  19. Unscheduled DNA synthesis in xeroderma pigmentosum cells after microinjection of yeast photoreactivating enzyme.

    NARCIS (Netherlands)

    J.C.M. Zwetsloot; J.H.J. Hoeijmakers (Jan); W. Vermeulen (Wim); A.P.M. Eker (André); D. Bootsma (Dirk)

    1986-01-01

    textabstractPhotoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers

  20. Gamma-ray induced inhibition of DNA synthesis in ataxia telangiectasia fibroblasts is a function of excision repair capacity

    International Nuclear Information System (INIS)

    Smith, P.J.; Paterson, M.C.

    1980-01-01

    The extent of the deficiency in γ-ray induced DNA repair synthesis in an ataxia telangiectasia (AT) human fibroblast strain was found to show no oxygen enhancement, consistent with a defect in the repair of base damage. Repair deficiency, but not repair proficiency, in AT cells was accompanied by a lack of inhibition of DNA synthesis by either γ-rays or the radiomimetic drug bleomycin. Experiments with 4-nitroquinoline 1-oxide indicated that lack of inhibition was specific for radiogenic-type damage. Thus excision repair, perhaps by DNA strand incision or chromatin modification, appears to halt replicon initiation in irradiated repair proficient cells whereas in repair defective AT strains this putatively important biological function is inoperative

  1. Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture.

    Science.gov (United States)

    Jhaveri, Ankur; Walsh, Stephen J; Wang, Yatzen; McCarthy, MaryBeth; Gronowicz, Gloria

    2008-11-01

    Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five-step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma-derived cell line, SaOs-2. No significant differences were found in DNA synthesis, assayed by [(3)H]-thymidine incorporation at 1 or 2 weeks for SaOs-2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (p = 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (p = 0.016) and decreased mineralization in SaOs-2 (p = 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT-induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs-2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma-derived cell line. (c) 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  2. Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants of Ustilago maydis

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.

    1977-01-01

    The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungus Ustilago maydis after incubation at the restrictive temperature (32/sup 0/C) for eight hours. Mutants ts-220, ts-207, ts-432 and ts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutants ts-20, tsd 1-1, ts-84 and pol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutant pol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutant ts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32/sup 0/C. tsd 1-1 and ts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis which correlates to increasing UV sensitivity of these strains on incubation at 32/sup 0/C. A pol 1-1 ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

  3. Peripheral SLC6A4 DNA methylation is associated with in vivo measures of human brain serotonin synthesis and childhood physical aggression.

    Directory of Open Access Journals (Sweden)

    Dongsha Wang

    Full Text Available The main challenge in addressing the role of DNA methylation in human behaviour is the fact that the brain is inaccessible to epigenetic analysis in living humans. Using positron emission tomography (PET measures of brain serotonin (5-HT synthesis, we found in a longitudinal sample that adult males with high childhood-limited aggression (C-LHPA had lower in vivo 5-HT synthesis in the orbitofrontal cortex (OBFC. Here we hypothesized that 5-HT alterations associated with childhood aggression were linked to differential DNA methylation of critical genes in the 5-HT pathway and these changes were also detectable in peripheral white blood cells. Using pyrosequencing, we determined the state of DNA methylation of SLC6A4 promoter in T cells and monocytes isolated from blood of cohort members (N = 25 who underwent a PET scan, and we examined whether methylation status in the blood is associated with in vivo brain 5-HT synthesis. Higher levels of methylation were observed in both T cells and monocytes at specific CpG sites in the C-LHPA group. DNA methylation of SLC6A4 in monocytes appears to be associated more reliably with group membership than T cells. In both cell types the methylation state of these CpGs was associated with lower in vivo measures of brain 5-HT synthesis in the left and right lateral OBFC (N = 20 where lower 5-HT synthesis in C-LHPA group was observed. Furthermore, in vitro methylation of the SLC6A4 promoter in a luciferase reporter construct suppresses its transcriptional activity supporting a functional role of DNA methylation in SLC6A4 promoter regulation. These findings indicate that state of SLC6A4 promoter methylation is altered in peripheral white blood cells of individuals with physical aggression during childhood. This supports the relevance of peripheral DNA methylation for brain function and suggests that peripheral SLC6A4 DNA methylation could be a marker of central 5-HT function.

  4. Fidelity of DNA Replication in Normal and Malignant Human Breast Cells.

    Science.gov (United States)

    1997-08-01

    enzyme) into the multiple cloning site (MCS). This template will not only replicate inside a mammalian cell (utilizing the E-B virus origin), and...Maniatis, T. Commonly used techniques in molecular cloning . In: Molecular cloning : REFERENCES a laboratory manual, 2nd edition. Cold Spring Harbor...A vatit"Y Of DNA synthesis and the typt of DNA replica~tion Products " celular prca including DNA rsplicatlon. DNA repsair. R~NA formed in experiments

  5. X-ray induced degradation of DNA in Aspergillus nidulans cells comparative analysis of UV- and X-ray induced DNA degradation

    International Nuclear Information System (INIS)

    Zinchenko, V.V.; Babykin, M.M.

    1980-01-01

    Irradiating cells of Aspergillus nidulans of the wild type in the logarythmical growth phase with X-rays leads to a certain retention in DNA synthesis. This period is characterized by an insignificant fermentative DNA degradation connected with a process of its repair. There is no direct dependence between the radiation dose and the level of DNA degradation. The investigation of X-ray induced DNA degradation in a number of UVS-mutants permits to show the existence of two branches of DNA degradation - dependent and independent of the exogenic energy source. The dependence of DNA degradation on albumen synthesis prior to irradiation and after it, is demonstrated. It is supposed that the level of X-ray induced DNA degradation is determined by two albumen systems, one of which initiates degradation and the other terminates it. The comparative analysis of UV and X-ray induced DNA degradation is carried out

  6. Second-strand cDNA synthesis: classical method

    International Nuclear Information System (INIS)

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

  7. Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

    International Nuclear Information System (INIS)

    Tong, P.; Mayes, D.; Wheeler, L.

    1986-01-01

    The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca ++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca ++ medium (0.09 mM) as measured by incorporation of [ 3 H] thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC 50 of about 10 μM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 μM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number of control at 10 μM. These results are of importance since they suggest Ca ++ may influence the effect of retinoids on keratinocytes

  8. Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

    International Nuclear Information System (INIS)

    Minagawa, T.; Nakaya, C.; Iida, H.

    1974-01-01

    Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)

  9. Damage of DNA and plasma membranes in murine lymphoma cells irradiated under aerobic or hypoxic conditions

    International Nuclear Information System (INIS)

    Wlodek, D.

    1983-01-01

    A review of the knowledge of radiation effects on cell membranes and DNA and of repair mechanisms of radiation lesions is given. Investigations of properties of plasma membranes in L5178Y-S and L5178Y-R cells (surface charge, fluidity, transport of amino acids) indicate that there is no direct connection between membrane lesions and reproductive death. It was also found that in irradiated cells of both L5178Y-strains the rate of DNA chain elongation is the same, similarly as the amount of the initial DNA lesions and the rate of repair processes. Difference in the level of DNA synthesis inhibition is not proportional to the lethal effect. The results are also reported point to the difference between L5178Y-S and L5178Y-R cells in susceptibility of post-irradiation DNA synthesis to factors modifying chromatin conformation, such as inhibitors of (ADP-ribose) n polymerase. 221 refs. (author)

  10. Postreplication gap filling in the DNA of X-ray-damaged Chinese hamster cells

    International Nuclear Information System (INIS)

    Koerner, I.; Malz, W.

    1975-01-01

    In X-irradiated Chinese hamster cells the newly synthesized DNA has a lower molecular weight than the DNA in control cells. This reduced molecular weight has been interpreted by gap induction opposite the lesions in the parental DNA strands. Within two hours these postreplication gaps were closed. With the aid of BrdUrd-photolys-technique it could be demonstrated that the gaps were filled by de novo synthesis. But we were not able to show a participation of parental DNA in the gap-filling process

  11. Influence of vinyl chloride monomer and vinyl chloride monomer derivatives on hepatic DNA synthesis

    International Nuclear Information System (INIS)

    Brenner, E.A.

    1982-01-01

    Vinyl chloride monomer (VCM) is used extensively in the chemical industry, mainly in the production of polyvinyl chloride. It has recently been found to cause hepatic angiosarcoma. As VCM has also been shown to be mutagenic after metabolic activation the effect of VCM on DNA synthesis was investigated. [ 3 H]Thymidine incorporation into DNA was used to measure the rate of DNA synthesis in regenerating rat liver. A possible direct toxic effect of VCM or its metabolites on liver cell metabolism was examined by two unrelated techniques, viz. the measurement of adenine nucleotide concentrations in regenerating livers and the influence on transmembrane potentials in hepatocytes. The distribution of radioactivity in subcellular fractions following [ 14 C]VCM administration suggested microsomal conversion of VCM to an active form which was selectively retained in the nuclear fraction. Measurement of the activities of thymidine kinase and DNA polymerase in regenerating liver indicated that the induction of these enzymes which normally occurs after partial hepatectomy was not prevented by VCM treatment. Three techniques were used to test the hypothesis that the retardation in DNA synthesis was due to DNA damage: the prophage lambda induction test for DNA damage, autoradiographic detection of unscheduled thymidine incorporation into DNA, and detection of DNA strand breaks in alkaline sucrose gradients. All three provided evidence of DNA damage and led to the development of a novel technique to confirm these findings. This involved centrifugation in neutral sucrose gradients on intact double-stranded DNA contained in hepatocyte nucleoids and showed conclusively that VCM administration causes DNA strand breaks. Subsequent repair of DNA was also assessed by this technique. The site of the VCM/metabolite: DNA reaction was characterized by DNA thermal denaturation and renaturation studies

  12. Correspondence: chromosomal localization of uv-induced unscheduled DNA synthesis

    International Nuclear Information System (INIS)

    Berliner, J.; Mello, R.S.; Norman, A.

    1976-01-01

    We have measured the grain density - the number of grains per unit length - over the centromere and noncentromere regions of metaphase chromosomes in autoradiographs of human lymphocytes. When the chromosomes were labeled in G 0 by uv-induced unscheduled DNA synthesis, the grain density was two to four times larger over the centromere than over the noncentromere regions. When the labeling was done by scheduled DNA synthesis in S or unscheduled synthesis in M, the grain densities were approximately equal over both regions

  13. Effect of haloperidol on the synthesis of DNA in the pituitary gland of the rat.

    Science.gov (United States)

    Machiavelli, G A; Jahn, G A; Kalbermann, L E; Szijan, I; Alonso, G E; Burdman, J A

    1982-03-01

    The administration of haloperidol increased serum prolactin and decreased the pituitary concentration of prolactin 15 min after its administration. Concomitantly there was a stimulation in the synthesis of DNA and the activity of DNA polymerase alpha in the anterior pituitary gland that was greater in oestrogenized than in non-oestrogenized male rats. Both these effects were greatly reduced by clomiphene in the oestrogenized male rats, although it did not affect the release of prolactin produced by haloperidol. In non-oestrogenized animals clomiphene abolished the stimulatory effect of haloperidol on the synthesis of DNA. These results suggest that the reduction in the intracellular levels of prolactin are a primary event in the oestrogen mediated stimulation of cell proliferation by prolactin releasing agents.

  14. DNA excision repair in permeable human fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells

  15. Lack of effect of inhibitors of DNA synthesis/repair on the ionizing radiation-induced chromosomal damage in G[sub 2] stage of ataxia telangiectasia cells

    Energy Technology Data Exchange (ETDEWEB)

    Antoccia, A. (Univ. ' La Sapienza' , Rome (Italy). Dipt. di Genetica e Biologia Molecolare); Palitti, F.; Raggi, T. (Univ. del Tuscia, Viterbo (Italy). Dipt. di Agrobiologia ed Agrochimica); Catena, C. (ENEA, Casaccia (Italy). Centro Ricerche Energia); Tanzarella, C. (Rome Univ. 3 (Italy). Dipt. di Biologia)

    1994-09-01

    The relationship between the repair processes occurring at the G[sub 2] phase of the cell cycle and cytogenetic damage in ataxia telangiectasia (AT) cells was studied. Lymphoblastoid cells derived from normal, heterozygote AT (HzAT) and three AT patients were exposed to X-rays or fission neutrons and post-treated with inhibitors of DNA synthesis/repair, such as inhibitors of DNA polymerases [alpha], [sigma] and [epsilon] (cytosine arabinoside, ara-C; aphidicolin, APC; buthylphenyl-guanine, BuPdG) or ribonucleotide reductase (hydroxyurea HU). A strong increase of radiation-induced chromosomal aberrations was observed in normal and HzAT cells post-treated with ara-C, APC and HU, but not in the presence of BuPdG. No enhancing effect was observed in cells derived from AT patients, except for HU post-irradiation treatment. These results suggest that the enzymes that can be inhibited by these agents are not directly involved in the repair of radiation damage induced in G[sub 2] cells from AT patients, indicating that probably the AT cells that we used lack the capability to transform the primary DNA lesions into reparable products, or that AT cells might contain a mutated form of DNA polymerase resistant to the inhibitors. (author).

  16. Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

    Science.gov (United States)

    Stengel, Gudrun; Kuchta, Robert D

    2011-01-01

    The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

  17. Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Victoria C. Foletta

    2016-10-01

    Full Text Available Deuterated water (2H2O, a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules, thus permitting the calculation of their synthesis rates. Here, we have combined 2H2O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation, protein and lipid synthesis rates. We have used this approach to characterize the turnover rates and metabolism of a panel of mammalian cells in vitro (muscle C2C12 and colon cancer cell lines. Our data show that in actively-proliferating cells, biomass synthesis rates are strongly linked to the rate of cell division. Furthermore, in both proliferating and non-proliferating cells, it is the lipid pool that undergoes the most rapid turnover when compared to DNA and protein. Finally, our data in human colon cancer cell lines reveal a marked heterogeneity in the reliance on the de novo lipogenic pathway, with the cells being dependent on both ‘self-made’ and exogenously-derived fatty acid.

  18. Effect of human cell malignancy on activity of DNA polymerase iota.

    Science.gov (United States)

    Kazakov, A A; Grishina, E E; Tarantul, V Z; Gening, L V

    2010-07-01

    An increased level of mutagenesis, partially caused by imbalanced activities of error prone DNA polymerases, is a key symptom of cell malignancy. To clarify the possible role of incorrect DNA polymerase iota (Pol iota) function in increased frequency of mutations in mammalian cells, the activity of this enzyme in extracts of cells of different mouse organs and human eye (melanoma) and eyelid (basal-cell skin carcinoma) tumor cells was studied. Both Mg2+, considered as the main activator of the enzyme reaction of in vivo DNA replication, and Mn2+, that activates homogeneous Pol iota preparations in experiments in vitro more efficiently compared to all other bivalent cations, were used as cofactors of the DNA polymerase reaction in these experiments. In the presence of Mg2+, the enzyme was active only in cell extracts of mouse testicles and brain, whereas in the presence of Mn2+ the activity of Pol iota was found in all studied normal mouse organs. It was found that in cell extracts of both types of malignant tumors (basal-cell carcinoma and melanoma) Pol iota activity was observed in the presence of either Mn2+ or Mg2+. Manganese ions activated Pol iota in both cases, though to a different extent. In the presence of Mn2+ the Pol iota activity in the basal-cell carcinoma exceeded 2.5-fold that in control cells (benign tumors from the same eyelid region). In extracts of melanoma cells in the presence of either cation, the level of the enzyme activity was approximately equal to that in extracts of cells of surrounding tumor-free tissues as well as in eyes removed after traumas. The distinctive feature of tissue malignancy (in basal-cell carcinoma and in melanoma) was the change in DNA synthesis revealed as Mn2+-activated continuation of DNA synthesis after incorrect incorporation of dG opposite dT in the template by Pol iota. Among cell extracts of different normal mouse organs, only those of testicles exhibited a similar feature. This similarity can be explained by

  19. Molecular Mechanisms of DNA Replication Checkpoint Activation

    Directory of Open Access Journals (Sweden)

    Bénédicte Recolin

    2014-03-01

    Full Text Available The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability.

  20. Interferon synthesis in mouse peritoneal cells damaged by x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Szolgay, E; T' alas, M

    1976-01-01

    NDV-induced interferon of peritoneal cells of irradiated (x-rays, 400 R) and control mice was investigated in vitro. Irradiation or treatment with hydroxyurea (10(-5) M) and mitomycin C (25 microng/ml) did not change interferon synthesis in spite of an 80 to 90% inhibition of 3H-thymidine incorporation. Increased doses of mitomycin C and treatment with actinomycin D and puromycin blocked interferon production. De novo interferon synthesis occurred in cells with damaged replicative activity of DNA caused by irradiation or by treatment with antimetabolites.

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

    Science.gov (United States)

    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.

  2. Involvement of sulfoquinovosyl diacylglycerol in DNA synthesis in Synechocystis sp. PCC 6803

    Directory of Open Access Journals (Sweden)

    Aoki Motohide

    2012-02-01

    Full Text Available Abstract Background Sulfoquinovosyl diacylglycerol (SQDG is present in the membranes of cyanobacteria and their postulated progeny, plastids, in plants. A cyanobacterium, Synechocystis sp. PCC 6803, requires SQDG for growth: its mutant (SD1 with the sqdB gene for SQDG synthesis disrupted can grow with external supplementation of SQDG. However, upon removal of SQDG from the medium, its growth is retarded, with a decrease in the cellular content of SQDG throughout cell division, and finally ceases. Concomitantly with the decrease in SQDG, the maximal activity of photosynthesis at high-light intensity is repressed by 40%. Findings We investigated effects of SQDG-defect on physiological aspects in Synechocystis with the use of SD1. SD1 cells defective in SQDG exhibited normal photosynthesis at low-light intensity as on culturing. Meanwhile, SD1 cells defective in SQDG were impaired in light-activated heterotrophic growth as well as in photoautotrophic growth. Flow cytometric analysis of the photoautotrophically growing cells gave similar cell size histograms for the wild type and SD1 supplemented with SQDG. However, the profile of SD1 defective in SQDG changed such that large part of the cell population was increased in size. Of particular interest was the microscopic observation that the mitotic index, i.e., population of dumbbell-like cells with a septum, increased from 14 to 29% in the SD1 culture without SQDG. Flow cytometric analysis also showed that the enlarged cells of SD1 defective in SQDG contained high levels of Chl, however, the DNA content was low. Conclusions Our experiments strongly support the idea that photosynthesis is not the limiting factor for the growth of SD1 defective in SQDG, and that SQDG is responsible for some physiologically fundamental process common to both photoautotrophic and light-activated heterotrophic growth. Our findings suggest that the SQDG-defect allows construction of the photosynthetic machinery at an

  3. Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Zhaohua PEng [Mississippi State University; Ronald, Palmela [UC-Davis; Wang, Guo-Liang [The Ohio State University

    2013-04-26

    This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for each gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall

  4. Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo

    International Nuclear Information System (INIS)

    Tanaka, K.; Hayakawa, H.; Sekiguchi, M.; Okada, Y.

    1977-01-01

    The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by uv light. A clear dose response was observed between the level of uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4v 1 , a mutant defective in the endonuclease V gene, showed no ability to restore the uv-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by uv light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after uv irradiation

  5. Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene

    International Nuclear Information System (INIS)

    Knight, G.B.; Gudas, J.M.; Pardee, A.B.

    1987-01-01

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, the authors have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene

  6. DNA-repair after irradiation of cells with gamma-rays and neutrons

    International Nuclear Information System (INIS)

    Altmann, H.

    1975-11-01

    The structural alterations of calf thymus DNA produced by neutron or gamma irradiation were observed by absorption spectra, sedimentation rate and viscosity measurements. Mixed neutron-gamma irradiation produced fewer single and double strand breaks compared with pure gamma irradiation. RBE-values for mixed neutron-gamma radiation were less than 1, and DNA damage decreased with increasing neutron dose rate. Repair processes of DNA occuring after irradiation were measured in mouse spleen suspensions and human lymphocytes using autoradiographic methods and gradient centrifugations. The number of labelled cells was smaller after mixed neutron-gamma irradiation than after gamma irradiation. The rejoining of strand breaks in alkaline and neutral sucrose was more efficient after gamma irradiation than after mixed neutron-gamma irradiation. Finally, the effect of detergents Tween 80 and Nonident P40 on unscheduled DNA synthesis was studied by autoradiography after mixed neutron-gamma irradiation (Dn=5 krad). The results showed that the DNA synthesis was inhibited by detergent solutions of 0.002%

  7. Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Morris, D R; Jorstad, C M; Seyfried, C E

    1977-09-01

    The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

  8. Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

    International Nuclear Information System (INIS)

    Stark, M.; Naiman, T.; Canaani, D.

    1989-01-01

    In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced [ 3 H]thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line

  9. Induced DNA repair pathway in mammalian cells

    International Nuclear Information System (INIS)

    Overberg, R.

    1985-01-01

    The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 μM cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells

  10. Molecular mechanisms of induced mutagenesis. Replication in vivo of bacteriophage phiX174 single-stranded, ultraviolet light-irradiated DNA in intact and irradiated host cells

    Energy Technology Data Exchange (ETDEWEB)

    Caillet-Fauquet, P; Defais, M; Radman, M [Brussels Univ. (Belgium)

    1977-11-25

    Genetic analysis has revealed that radiation and many chemical mutagens induce in bacteria an error-prone DNA repair process which is responsible for their mutagenic effect. The biochemical mechanism of this inducible error-prone repair has been studied by analysis of the first round of DNA synthesis on ultraviolet light-irradiated phiX174 DNA in both intact and ultraviolet light-irradiated host cells. Intracellular phiX174 DNA was extracted, subjected to isopycnic CsCl density-gradient analysis, hydroxylapatite chromatography and digestion by single-strand-specific endonuclease S/sub 1/. Ultraviolet light-induced photolesions in viral DNA cause a permanent blockage of DNA synthesis in intact Escherichia coli cells. However, when host cells were irradiated and incubated to induce fully the error-prone repair system, a significant fraction of irradiated phiX174 DNA molecules can be fully replicated. Thus, inducible error-prone repair in E.coli is manifested by an increased capacity for DNA synthesis on damaged phiX174 DNA. Chloramphenicol (100 ..mu.. g/ml), which is an inhibitor of the inducible error-prone DNA repair, is also an inhibitor of this particular inducible DNA synthesis.

  11. Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

    International Nuclear Information System (INIS)

    Zwelling, L.A.; Kerrigan, D.; Mattern, M.R.

    1983-01-01

    The synthesis of poly(adenosine diphosphoribose [poly(ADP-R)] follows the DNA strand breakage produced by a number of physical and chemical agents, including X-radiation, and may be important for repair of several types of DNA damage. The reduction or abolition of its synthesis following X-irradiation might explain the enhanced sensitivity of ataxia-telangiectasia (A-T) cells to X-ray. We have examined 8 lines of human fibroblasts (including 4 A-T lines) for stimulation of the synthesis of poly(ADP-R) by X-irradiation. Similar amounts of X-ray-stimulated synthesis of poly(ADP-R) were detected in 4 lines of A-T fibroblasts, and in fibrolasts from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient and 2 normal patients. 6 lines of human lymphoblastoid lines were also examined for X-ray-stimulated poly(ADP-R) synthesis. 4 A-T lines displayed an unusually high synthesis of poly(ADP-R) in unirradiated cells compared with 2 normal lines. (orig./AJ)

  12. Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Zwelling, L.A.; Kerrigan, D. (National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Pharmacology); Mattern, M.R. (National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Carcinogenesis)

    1983-04-01

    The synthesis of poly(adenosine diphosphoribose (poly(ADP-R)) follows the DNA strand breakage produced by a number of physical and chemical agents, including X-radiation, and may be important for repair of several types of DNA damage. The reduction or abolition of its synthesis following X-irradiation might explain the enhanced sensitivity of ataxia-telangiectasia (A-T) cells to X-ray. We have examined 8 lines of human fibroblasts (including 4 A-T lines) for stimulation of the synthesis of poly(ADP-R) by X-irradiation. Similar amounts of X-ray-stimulated synthesis of poly(ADP-R) were detected in 4 lines of A-T fibroblasts, and in fibrolasts from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient and 2 normal patients. 6 lines of human lymphoblastoid lines were also examined for X-ray-stimulated poly(ADP-R) synthesis. 4 A-T lines displayed an unusually high synthesis of poly(ADP-R) in unirradiated cells compared with 2 normal lines.

  13. DNA repair in human cells: Methods for the determination of calmodulin involvement

    International Nuclear Information System (INIS)

    Charp, P.A.

    1987-01-01

    Exposure of DNA to either physical or chemical agents can result in the formation of a number of different lesions which must be repaired enzymatically in order for DNA to carry on normal replication and transcription. In most cases, the enzymes involved in this repair of damaged DNA include endonucleases, exonucleases, glycosylases, polymerases, and ligases. Each group of enzymes is involved in precise steps in DNA repair. Exposure to physical agents such as ultraviolet light (UV) at a wavelength of 254 nm is repaired by two distinct and different mechanisms. One mode of enzymatic repair of pyrimidine dimers is accomplished in situ by photoreactivation of UV-induced pyrimidine dimers by photoreactivating light. The second mode of enzymatic repair is the excision repair of pyrimidine dimers involving several different enzymes including endonuclease, exonuclease, and DNA ligase. A summary of the sequence of enzymatic steps involved is shown. It has been observed that specific drugs which bind to and alter the action of calmodulin in cells block DNA synthesis. This suggests that calmodulin may play a role both in normal DNA replication and repair. Others using an indirect method measuring the degree of DNA nucleoid sedimentation, showed that the specific anti-calmodulin agent W-13 slowed the rate of DNA repair. Others showed that DNA synthesis in T51B rat liver cells could be blocked with the addition of either chlorpromazine or trifluoperazine

  14. Relationship of radiation sensitivity and aberrant DNA synthesis in repair deficient CHO cells

    International Nuclear Information System (INIS)

    Newman, C.N.; Hagler, H.; Miller, J.H.

    1986-11-01

    Comparison of alkaline sucrose gradient profiles of pulse-labeled DNA from a normal CHO cell line and its radiation-sensitive mutant, xrs-5, reveals significant differences in the replicon elongation/maturation process in these two cells. During a one hr period of growth subsequent to labeling, the molecular weight of pulse-labeled DNA from the mutant cell increases considerably more rapidly than that of the parent cell. For xrs-5, the presence of 2 mM deoxycytidine (CdR) in the culture medium reduces the replication rate to one approaching that of the parent cell growing in the standard medium. Corresponding uv resistance of the mutant likewise increases to nearly that of the parent cell line. These results suggest that the locus conferring radiation sensitivity to xrs-5 affects the DNA replisome complex and that replicative activity and radiation sensitivity are jointly modulated by CdR. 19 refs., 4 figs

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  16. Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

    DEFF Research Database (Denmark)

    Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe

    2011-01-01

    We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilizati...

  17. UAP56 is an important mediator of Angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation

    International Nuclear Information System (INIS)

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey

    2013-01-01

    Highlights: ► Knockdown of UAP56 inhibits Angiotensin II/PDGF induced vascular smooth muscle cell proliferation. ► UAP56 is a positive regulator of E2F transcriptional activation. ► UAP56 is present in the vessel wall of low flow carotid arteries. -- Abstract: Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease

  18. Stimulatory effect of low dose radionuclide on DNA synthesis and UDS in splenic lymphocytes

    International Nuclear Information System (INIS)

    Zhu Shoupeng; Yang Zhanshan

    1999-12-01

    To study the stimulatory effect on DNA synthesis and unscheduled DNA synthesis (UDS) in splenic lymphocytes induced by low dose enriched uranium 235 U. By using 3 H-TdR incorporation assay technique, the DNA replicative synthesis in PHA and LPS stimulated splenic lymphocytes was observed. By using DNA synthesis inhibitor such as hydroxyurea, the UV-induced unscheduled DNA synthesis in splenic lymphocytes occurred. When the injected low dose of enriched uranium 235 u was 0.1 μg/kg body weight, the transformation capacity was elevated for splenic T lymphocytes, simultaneously the stimulative index increased. The UDS of splenic lymphocytes induced by ultra-violate revealed a statistically significant increase by low dose of enriched uranium 235 U at the range of 0.1-20 μg/kg body weight. A stimulatory action of low dose enriched uranium 235 U on DNA replicative synthesis as well as on UV-induced UDS in splenic lymphocytes was detected

  19. Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging

    International Nuclear Information System (INIS)

    Nagata, Tetsuji

    2010-01-01

    Study aging changes of intramitochondrial DNA, RNA, protein synthesis of mouse organs during the development and aging, 30 groups of developing and aging mice (3 individuals each), from fetal day 19 to postnatal newborn at day 1, 3, 9, 14 and adult at month 1, 2, 6, 12 to 24, were injected with either 3 H-thymidine, 3 H-uriidine, or 3 H-leucine, sacrificed 1 h later and liver, adrenal, lung and testis tissues observed by electron microscopic radioautography. Accordingly, numbers of mitochondria per cell profile area, numbers of labeled mitochondria and the mitochondrial labeling index labeled with 3 H-labeled precursors showing DNA, RNA, protein synthesis in these cells (hepatocytes, 3 zones of the adrenal cortices - zona glomerulosa, fasciculata and reticularis -, adrenal medullary cells, pulmonary cells and testis cells) were counted per cells and compared among the respective developing and aging groups. The numbers of mitochondria in these cells increased from fetal day 19 to postnatal month 1 and 2. However, the numbers of labeled mitochondria and the labeling indices of intramitochondrial DNA, RNA, protein syntheses incorporating the 3 H-labeled precursors in the described tissue cells increased from fetal day 19 to postnatal month 1 and decreased to month 24. These data support that the activity of intramitochnodrial DNA, RNA, protein syntheses in cells of these tissues increased and decreased by development and aging in mice. The intramitochondrial DNA, RNA and protein syntheses in some other organs were also reviewed and discussed. (author)

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

    Kim, Sunyoung; Baltimore, D.; Byrn, R.; Groopman, J.

    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. The authors 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

  2. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  3. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    International Nuclear Information System (INIS)

    Nolan, N.L.; Kidwell, W.R.

    1982-01-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37 0 C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of γ-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of γ-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels

  4. Analysis of translesion DNA synthesis activity of the human REV1 protein, which is a key player in radiation-induced mutagenesis

    International Nuclear Information System (INIS)

    Masuda, Yuji; Kamiya, Kenji

    2003-01-01

    Ionizing radiation frequently causes oxidative DNA damage in cells. It has been suggested that functions of the REV1 gene are induction of mutations and prevention of cell death caused by ionizing radiation through the damage bypass DNA replication. The gene product possesses a deoxycytidyl transferase activity, which is required for translesion DNA synthesis of a variety of damaged bases and an abasic site. To elucidate molecular mechanisms of the mutagenesis and translesion DNA synthesis, it is important to characterize the enzymatic properties of the REV1 protein. Here, we describe a novel method for purifying the recombinant human REV1 protein and the anzymatic properties of the protein. We established an efficient system for induction of the recombinant human REV1 protein in Escherichia coli cells. The REV1 protein was purified to homogeneity using nickel-chelating sepharose, heparin sepharose and superdex 200 chromatography. When purified by this method, REV1 protein is free of endo-, exonuclease and DNA polymerase activities. The purified REV1 protein is suitable for enzymological studies, and we used this to biochemical characterization. The REV1 protein inserts dCMP opposite templates G, A, T, C and an abasic site and inserts dGMP and dTMP opposite template G. Kinetic analysis provided evidence for high efficiency for dCMP insertion opposite template G and an abasic site, suggesting that the REV1 protein play a role in translesion DNA synthesis of an abasic site. (author)

  5. DNA synthesis in the imaginal wing discs of the American bollworm ...

    Indian Academy of Sciences (India)

    Unknown

    The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids. 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instar Helicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of.

  6. Excision of pyrimidine dimers from epidermal DNA and nonsemiconservative epidermal DNA synthesis following ultraviolet irradiation of mouse skin

    International Nuclear Information System (INIS)

    Bowden, G.T.; Trosko, J.E.; Shapas, B.G.; Boutwell, R.K.

    1975-01-01

    Pyrimidine dimer production and excision in epidermal DNA were studied at five different dose levels of ultraviolet light in the skin of intact mice. Dimer production increased with dose up to 50,400 ergs/sq mm. Approximately 30 percent of the thymine-containing dimers were excised by 24 hr after irradiation at three lower dose levels of ultraviolet light. Nonsemiconservative DNA replication in ultraviolet-irradiated mouse skin was shown to continue for at least 18 hr. The rate of nonsemiconservative replication decreased with time, but did so slowly. The initial rates of nonsemiconservative replication increased with ultraviolet light dose levels up to about 4200 ergs/sq mm, after which the initial rates were decreased. Semiconservative epidermal DNA synthesis was shown to be inhibited by hydroxyurea, but hydroxyurea had no effect on ultraviolet light-induced nonsemiconservative DNA replication. The observed pyrimidine dimer excision and nonsemiconservative DNA replication suggest that in the intact mouse the cells of the epidermis are capable of DNA excision repair after ultraviolet irradiation of mouse skin

  7. Nuclear DNA synthesis rate and labelling index: effects of carcinogenic and non-carcinogenic chemicals on its behaviour in the organism of growing CBA mice

    International Nuclear Information System (INIS)

    Amlacher, E.; Rudolph, C.

    1978-01-01

    Well known bioassays have been compared with the author's thymidine incorporation-screening system and other assays based on biochemical quantification of DNA synthesis as a possibility of identification of carcinogens. The partial inhibition of the whole DNA synthesis in a proliferating cell population after treatment with toxic and carcinogenic chemicals is an early common response especially in hepatectomized animal, livers caused by the effects of those substances. However, by quantitative evaluation of the nuclear DNA synthesis rate as a basic parameter, using autoradiographs of kidney and liver of juvenile growing CBA mice, it is possible to differentiate carcinogenic from non-carcinogenic chemicals by means of silver grain counting after 3 H-TdR incorporation. On the contrary, the whole DNA synthesis, expressed by the 3 H-labelling index (in per cent) of kidney and liver, did not permit such a differentiation in the experimental arrangement used. It could be demonstrated that carcinogenic compounds of different chemical classes partially inhibit the nuclear DNA synthesis rate significantly over a period of more than 24 hours. The tested non-carcinogenic compounds did not show this suppressive effect on the nuclear DNA synthesis rate. (author)

  8. Absence of specificity in inhibition of DNA repair replication by DNA-binding agents, cocarcinogens, and steroids in human cells

    International Nuclear Information System (INIS)

    Cleaver, J.E.; Painter, R.B.

    1975-01-01

    Although many chemicals, including cocarcinogens, DNA-binding agents, and steroids, inhibit repair replication of ultraviolet-induced damage to DNA in human lymphocytes and proliferating cells in culture, none of these chemicals is specific. Our results show that all the chemicals we tested inhibit normal DNA synthesis as much as or more than they inhibit repair replication. There is thus no evidence in our results to support the hypothesis that cocarcinogens are specific inhibitors of DNA repair or that any of the chemicals studied might be useful adjuncts to tumor therapy merely because of specific inhibition of radiation repair mechanisms

  9. Repair of DNA in xeroderma pigmentosum conjunctiva

    International Nuclear Information System (INIS)

    Newsome, D.A.; Kraemer, K.H.; Robbins, J.H.

    1975-01-01

    Xeroderma pigmentosum (XP) is an autosomal recessive disease with tumor formation on sun-exposed areas of the skin and eyes. Cells from most XP patients are deficient in repairing DNA damaged by ultraviolet (uv) light as shown by a reduced rate of tritiated thymidine (3HTdR) incorporation during their DNA repair synthesis. We have studied such repair synthesis in conjunctival cells from an XP patient with a conjunctival epithelioma and from normal cadaver conjunctiva. Cultured conjunctival cells were irradiated with uv light and then incubated with 3HTdR. Autoradiograms were prepared and showed that uv radiation induced a considerably slower rate of DNA repair synthesis in the XP cells than in normal cells. Many of the ocular abnormalities of XP, including tumor formation, may be the result of this defective DNA repair process

  10. Degradation and repair of DNA from rat hepatoma cells after treatments with γ-rays and N-methyl-N-nitrosourea

    International Nuclear Information System (INIS)

    Zakrzhevskaya, D.G.; Kulagina, T.P.; Petrov, S.I.; Fomenko, L.A.; Gaziev, A.I.

    1977-01-01

    It has been shown, that DNA single-strand breaks induced in the cells of ascite hepatoma with γ-rays and metylnitrosourea (MNM) are effectively repaired. DNA two-strand breaks of hepatoma cells, treated with MNM are effectively repaired in situ as well. Only insignificant part of two-strand gamma-induced breaks in DNA of these cells is repaired during postirradiation period. Under combined effect of gamma rays and MNM on hepatoma cells a delay of DNA reparation and its further degradation as well as inhibition of nonplanned DNA synthesis and the suppression of DNA-polymerase 1 activity are observed

  11. Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

    International Nuclear Information System (INIS)

    Fritz, E.

    1994-06-01

    In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.) [de

  12. TopBP1-mediated DNA processing during mitosis.

    Science.gov (United States)

    Gallina, Irene; Christiansen, Signe Korbo; Pedersen, Rune Troelsgaard; Lisby, Michael; Oestergaard, Vibe H

    2016-01-01

    Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

  13. An assay system for factors involved in mammalian DNA replication

    International Nuclear Information System (INIS)

    Reinhard, P.; Maillart, P.; Schluchter, M.; Gautschi, J.R.; Schindler, R.

    1979-01-01

    An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [ 3 H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation.) (Auth.)

  14. Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Tetsuji, E-mail: nagata@kowagakuen.ac.j [Shinshu Univ. School of Medicine, Matsumoto (Japan). Dept. of Anatomy and Cell Biology

    2010-07-01

    Study aging changes of intramitochondrial DNA, RNA, protein synthesis of mouse organs during the development and aging, 30 groups of developing and aging mice (3 individuals each), from fetal day 19 to postnatal newborn at day 1, 3, 9, 14 and adult at month 1, 2, 6, 12 to 24, were injected with either {sup 3}H-thymidine, {sup 3}H-uriidine, or {sup 3}H-leucine, sacrificed 1 h later and liver, adrenal, lung and testis tissues observed by electron microscopic radioautography. Accordingly, numbers of mitochondria per cell profile area, numbers of labeled mitochondria and the mitochondrial labeling index labeled with {sup 3}H-labeled precursors showing DNA, RNA, protein synthesis in these cells (hepatocytes, 3 zones of the adrenal cortices - zona glomerulosa, fasciculata and reticularis -, adrenal medullary cells, pulmonary cells and testis cells) were counted per cells and compared among the respective developing and aging groups. The numbers of mitochondria in these cells increased from fetal day 19 to postnatal month 1 and 2. However, the numbers of labeled mitochondria and the labeling indices of intramitochondrial DNA, RNA, protein syntheses incorporating the {sup 3}H-labeled precursors in the described tissue cells increased from fetal day 19 to postnatal month 1 and decreased to month 24. These data support that the activity of intramitochnodrial DNA, RNA, protein syntheses in cells of these tissues increased and decreased by development and aging in mice. The intramitochondrial DNA, RNA and protein syntheses in some other organs were also reviewed and discussed. (author)

  15. The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo.

    Science.gov (United States)

    Capmany, G; Taylor, A; Braude, P R; Bolton, V N

    1996-05-01

    The timing of pronuclear formation and breakdown, DNA synthesis and cleavage during the first cell cycle of human embryogenesis are described. Pronuclei formed between 3 and 10 h post-insemination (hpi; median 8 hpi). S-phase commenced between 8 and 14 hpi, and was completed between 10 and 18 hpi. M-phase was observed between 22 and 31 hpi (median duration 3 h), and cleavage to the 2-cell stage took place between 25 and 33 hpi. The timing of the same events was determined in 1-cell embryos derived from re-inseminated human oocytes that had failed to fertilize during therapeutic in-vitro fertilization (IVF). In these embryos, pronuclei formed between 3 and 8 h post-re-insemination (hpr-i), coinciding with the beginning of S-phase. While S-phase was completed as early as 10 hpr-i in some embryos, it extended until at least 16 hpr-i in others. Pronuclear breakdown and cleavage occurred from 23 and 26 hpr-i respectively; however, they did not occur in some embryos until after 46 hpr-i. The results demonstrate a markedly greater degree of variation in the timing of these events in embryos derived from re-inseminated oocytes compared with embryos derived from conventional IVF, and thus throw into question the validity of using the former as models for studies of the first cell cycle of human embryogenesis.

  16. Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

    Directory of Open Access Journals (Sweden)

    Hansen Peter J

    2008-01-01

    Full Text Available Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS, inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3 cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 μg/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

  17. Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements

    Directory of Open Access Journals (Sweden)

    Modesto Redrejo-Rodríguez

    2017-11-01

    Full Text Available Family B DNA polymerases (PolBs play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB, that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.

  18. B cells in the appendix and other lymphoid organs of the rabbit: stimulation of DNA synthesis by anti-immunoglobulin

    International Nuclear Information System (INIS)

    Calkins, C.E.; Ozer, H.; Waksman, B.H.

    1975-01-01

    Lymphocytes from rabbit lymphoid organs were cultured in the presence of class specific anti-immunoglobulin sera and of anti-allotype sera. Stimulation of tritiated thymidine uptake into DNA was taken to indicate the presence of the corresponding immunoglobulins on the cell surfaces. Thymus and bone marrow lymphocytes were unresponsive to all reagents tested. Popliteal lymph node contained cells responsive to anti-μ, anti-γ, and anti-α and therefore presumably bearing IgM, IgG, and IgA. Spleen had only IgM- and IgG-bearing cells, and the appendix contained cells with IgM and IgA receptors only. The lymph node, spleen, and appendix cells of rabbits depleted of B lymphocytes by irradiation (900 R) and injection of thymocytes were unresponsive to anti-immunoglobulin but were stimulated at almost normal levels by PHA and Con A. T cell-depleted animals (thymectomy, irradiation with three divided doses of 450 R and bone marrow shielding) had immunoglobulin-bearing lymphocytes but were unresponsive to the mitogens. However a moderate level of mitogen-responsiveness reappeared by 3 to 4 wk after irradiation. Cells of morphologically distinct regions of the appendix, separated manually, showed different responses corresponding to the inferred origins of these anatomic areas. The ''dome'' and ''corona'' contained functional IgM- and IgA-bearing cells. The ''TDA'' reacted well to PHA, Con A, and PWM, but was depleted of immunoglobulin-bearing cells. The ''follicle'' cells, which are almost all in active DNA synthesis or mitosis, were relatively unresponsive to either T or B cell stimuli. Anti-allotype serum stimulated the same populations which responded to class-specific heteroantisera but at a slightly lower level. It was inferred that gut-associated lymphoid tissues like the appendix may play a special role as an amplification site for B-cells destined to produce IgM and IgA elsewhere in the organism

  19. Efficiency and Fidelity of Human DNA Polymerases λ and β during Gap-Filling DNA Synthesis

    Science.gov (United States)

    Brown, Jessica A.; Pack, Lindsey R.; Sanman, Laura E.; Suo, Zucai

    2010-01-01

    The base excision repair (BER) pathway coordinates the replacement of 1 to 10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1- to 10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5′-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER. PMID:20961817

  20. Inhibition of DNA replication by ultraviolet light

    International Nuclear Information System (INIS)

    Edenberg, H.J.

    1976-01-01

    DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10 J/m 2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers

  1. Some new methyl-8-methoxypsoralens: synthesis, photobinding to DNA, photobiological properties and molecular modelling.

    Science.gov (United States)

    Gia, O; Anselmo, A; Pozzan, A; Antonello, C; Magno, S M; Uriarte, E

    1997-01-01

    The tricyclic structure of known natural photochemotherapeutic drugs such as 8-methoxypsoralen and 5-methoxypsoralen is often taken as a model in the search of new photosensitizer agents with less phototoxic and mutagenic effects. This paper describes the synthesis, characterization, photobinding to DNA, photobiological properties and computational chemistry of some 8-methoxypsoralen derivatives bearing two or three methyl groups at the key positions of the two photoactive double bonds. Results showed that photoreactivity and photobiological behaviour depend on the pattern of methyl substitutions. Antiproliferative activity in cell lines shows good correlation with DNA interaction data.

  2. DNA synthesis time in germinating rice and pattern of diethylsulphate induced mutations in pre-soaked seeds

    International Nuclear Information System (INIS)

    Narahari, P.

    1978-01-01

    DNA synthesis pattern in germinating rice seeds, pre-soaked in water for varying periods upto 48 hr, was determined by following the pulse incorporation of 3 H-thymidine into the TCA-insoluble nucleoprotein. Synthesis of DNA commenced at 24 hr, progressively increased to a first peak at about 38 hr, thereafter showed a 1/3rd drop and subsequently increased to a 2nd and still higher peak at 46 to 48 hr of pre-soaking. Treatments of diethylsulphate (dES) at a low concentration (0.2%-2hr) administered at various progressing stages of DNA synthesis resulted in decrease in seedling height and survival, and increase in mutation frequency at 45 hr. pre-soaking, maximum mutation frequencies of 20, 10 and 2% on M 1 plants, M 1 spikes and M 2 seedling bases, respectively were observed. Higher dES concentration (0.3%-2hr) given at later periods of pre-soaking showed near lethal effects and consequently decreased mutation frequencies. Treatments of sodium fluoride given singly or in combination with dES did not show any substantially different results as compared to those of the respective controls. Mutation spectra observed after dES treatments to germinating seeds, at different pre-soaking periods, were quite dissimilar. Specific mutations of economic importance like semi-dwarf mutants were isolated from the treatment of germinating seeds pre-soaked for 37.5 hr or more when shoot apex cells were undergoing DNA synthesis. (author)

  3. Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

    Science.gov (United States)

    Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

    2012-03-13

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

  4. Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    Science.gov (United States)

    Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2012-01-01

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

  5. Hydroxyurea does not prevent synchronized G1 Chinese hamster cells from entering the DNA synthetic period

    International Nuclear Information System (INIS)

    Walters, R.A.; Tobey, R.A.; Hildebrand, C.E.

    1976-01-01

    Using very high concentrations of radioactively labeled thymidine, we show that synchronized G 1 cells treated with hydroxyurea entered the DNA synthetic period at a time and rate indistinguishable from that of untreated cells, although the rate of DNA synthesis was greatly reduced in the drug-treated cultures. The DNA synthesized in the presence of hydroxyurea was less than or equal to 1 x 10 7 daltons, all of which could be chased into bulk DNA of approximately 3.5 x 10 8 daltons within 3 hr after removal of hydroxyurea. Hydroxyurea synchronized cells are apparently not blocked at the G 1 /S boundary but in the S phase itself

  6. Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    Directory of Open Access Journals (Sweden)

    Stougaard Magnus

    2007-11-01

    Full Text Available Abstract Background In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. Methods Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. Results An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1 on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. Conclusion Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as

  7. Failure of RNA synthesis to recover after UV irradiation: an early defect in cells from individuals with Cockayne's syndrome and xeroderma pigmentosum

    International Nuclear Information System (INIS)

    Mayne, L.V.; Lehmann, A.R.

    1982-01-01

    Previous work has shown that in cells from the ultraviolet-sensitive genetic disorder, Cockayne's syndrome, DNA synthesis fails to recover after ultraviolet irradiation, despite the fact that these cells have no detectable defect in either excision or daughter-strand repair pathways. We now show that Cockayne cells, as well as cells from a number of patients with xeroderma pigmentosum, are sensitive to the lethal effects of UV irradiation in stationary phase under conditions in which no DNA is synthesized after irradiation. Furthermore, in normal and defective human fibroblasts, RNA synthesis is depressed after UV irradiation. In normal (dividing) cells, RNA synthesis recovers very rapidly, but this recovery does not occur in Cockayne cells, and it is reduced or absent in xeroderma pigmentosum cells from different complementation groups. Qualitatively, similar results are obtained with cells in stationary phase. The recovery of RNA synthesis in the various defective cell strains is not correlated with the overall extent of excision repair, but there is some correlation between recovery of RNA synthesis and cell survival after ultraviolet irradiation. These results implicate recovery of RNA synthesis as an important early response to ultraviolet irradiation

  8. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Wang, Z.; Wu, X.; Friedberg, E.C.

    1993-01-01

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg 2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  9. Repair of soft X-ray damage to mammalian cell DNA

    Energy Technology Data Exchange (ETDEWEB)

    Meldrum, R.A.; Wharton, C.W. (Birmingham Univ. (UK). Dept. of Biochemistry)

    1990-10-01

    Inhibitors of polymerase {alpha} (hydroxyurea and cytosine arabinoside) and an inhibitor of polymerase {beta} and ''delta (di-deoxythymidine) had equal inhibitory effects on repair synthesis in the first 15 min after irradiation of Chinese hamster ovary cells with soft x-rays produced from a laser plasma. Polymerase {alpha} inhibitors had considerably more effect after 15 min following irradiation. This implies that polymerase {alpha}, {beta}, and/or {delta} are all equally active in the initial stages of repair synthesis after soft X-radiation, but {alpha}-activity is more prominent in later stages of repair synthesis. Polymerase {alpha} is thought to catalyse long-patch repair synthesis, while polymerase {beta} is thought to catalyse short-patch repair. Polymerase {delta} has been shown to be active in DNA repair synthesis, but its precise function is as yet uncertain. (author).

  10. Deficit in DNA content relative to histones in X-irradiated HeLa cells

    International Nuclear Information System (INIS)

    Bases, R.; Mendez, F.; Neubort, S.

    1976-01-01

    The DNA and histone content of HeLa S-3 cell cultures was measured by direct mass assays 21 hours after 1000 rad of X-irradiation, when the cells were arrested in G2 phase. The nuclear DNA content of such cultures was found to be deficient (73 per cent of control values). In contrast, the synthesis of nuclear histones persisted, and the total histone content was close to 100 per cent of control values. When synchronously-growing cultures were irradiated in mid-S phase and examined 3.5 hours later in G2 phase, both DNA and histone content were equal to control values. (author)

  11. An autoradiographic study of the synthesis of nucleic acids and protein during the cell cycle of synchronously dividing antheridial filaments in Chara vulgaris L

    Energy Technology Data Exchange (ETDEWEB)

    Olszewska, M J; Godlewski, M [Lodz Univ. (Poland)

    1972-01-01

    The synthesis of DNA, RNA, and protein in successive mitotic cycles of the synchronously dividing antheridial filaments of Chara vulgaris was studied autoradiographically. In all the generations examined, which enter the next mitosis, i.e., in the 4-, 8-, 16-, and 32-cell generations, the synthesis of DNA begins as early as telephase and continues into the early stages of interphase. The telephase cells of the 32-cell filaments do not incorporate //sup 3/H/thymidine, because the cells which arise from them do not divide but are transformed into spermatozoa. The DNA synthesis is accompanied by intense synthesis of RNA. The intensity of radioactivity calculated for 100 ..mu../sup 2/ of the area of the nucleus and cytoplasm is similar in all the generations, whereas the radioactivity induced by the incorporation of /8-/sup 14/C/adenine and//sup 3/H/phenylalanine calculated for one cell decreases proportionally to the reduction of the volume of the cytoplasm and nucleus in successive generations. (auth)

  12. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  13. DNA-cell conjugates

    Science.gov (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2018-05-15

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  14. Survey of current trends in DNA synthesis core facilities.

    Science.gov (United States)

    Hager, K M; Fox, J W; Gunthorpe, M; Lilley, K S; Yeung, A

    1999-12-01

    The Nucleic Acids Research Group of the Association of Biomolecular Resource Facilities (ABRF) last surveyed DNA synthesis core facilities in April 1995. Because of the introduction of new technologies and dramatic changes in the market, we sought to update survey information and to determine how academic facilities responded to the challenge presented by commercial counterparts. The online survey was opened in January 1999 by notifying members and subscribers to the ABRF electronic discussion group. The survey consisted of five parts: general facility information, oligonucleotide production profile, oligonucleotide charges, synthesis protocols, and trends in DNA synthesis (including individual comments). All submitted data were anonymously coded. Respondents from DNA synthesis facilities were primarily from the academic category and were established between 1984 and 1991. Typically, a facility provides additional services such as DNA sequencing and has upgraded to electronic ordering. There is stability in staffing profiles for these facilities in that the total number of employees is relatively unchanged, the tenure for staff averages 5.9 years, and experience is extensive. On average, academic facilities annually produce approximately 1/16 the number of oligonucleotides produced by the average commercial facilities, but all facilities report an increase in demand. Charges for standard oligonucleotides from academic facilities are relatively higher than from commercial companies; however, the opposite is true for modified phosphoramidites. Subsidized facilities charge less than nonsubsidized facilities. Synthesis protocols and reagents are standard across the categories. Most facilities offer typical modifications such as biotinylation. Despite the competition by large commercial facilities that have reduced costs dramatically, academic facilities remain a stable entity. Academic facilities enhance the quality of service by focusing on nonstandard

  15. DNA-synthesis of lymphocytes in hyperthyroid and enthyroid subjects. Effect of 131I therapy on hyperthyroidism

    International Nuclear Information System (INIS)

    Lundell, G.; Wasserman, J.; Einhorn, N.; Granberg, P.-O.

    1976-01-01

    The DNA-synthesis of human lymphoid cells as estimated by the measurement of thymidine incorporation in vitro was investigated in healthy controls and in patients with various thyroid disorders before and after therapy. Hyperthyroid patients treated with 131 I and surgery (euthyroid at initial blood sampling before surgery), patients with atoxic nodular goitre treated by surgery and healthy untreated control individuals comprised the material. The synthesis of DNA in lymphocytes was higher in hyperthyroid patients in comparison with euthyroid individuals, and decreased subsequent to 131 I therapy in the hyperthyroid patients. No decrease was recorded in the other groups of patients. No evidence suggesting a change in the lymphocyte reactivity to thyroglobulin was found in any of the patient groups. (author)

  16. 8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis

    DEFF Research Database (Denmark)

    Fengquin, X; Nielsen, Henrik; Zhen, W

    1993-01-01

    between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally......, it is shown that the cross-links in the rDNA molecules are repaired at equal rate in all three domains within 24 h and that RNA synthesis is partly restored during this repair period. The majority of the cells also go through one to two cell divisions in this period but do not survive....

  17. Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries

    Directory of Open Access Journals (Sweden)

    Ueno Shingo

    2004-09-01

    Full Text Available Abstract Background We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis. Results A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 1016 diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons. Conclusions Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases.

  18. Correlation between LH secretion in castrated rats with cellular proliferation and synthesis of DNA in the anterior pituitary gland.

    Science.gov (United States)

    Romano, M I; Machiavelli, G A; Pérez, R L; Carricarte, V; Burdman, J A

    1984-07-01

    The relationship between the release of LH and the synthesis of DNA was studied in the anterior pituitary gland of castrated rats. Cell types were characterized immunocytochemically. Castration significantly (P less than 0.01) increased the concentration of LH in serum (1326%) and the incorporation of [3H]thymidine into pituitary DNA (72%). This was accompanied by an increment in the activity of the enzyme DNA polymerase-alpha (58%) and in the number of mitoses (from 2 +/- 0.1/mm2 in intact rats to 21 +/- 0.8/mm2 15 days after castration). Only 20% of the mitoses found in the pituitary gland of castrated rats were positively stained with the antiserum against the beta-subunit of LH. The other 80% did not stain either with LH antiserum or with antisera against the other pituitary hormones. There was a significant (P less than 0.01) increase in the number of LH cells in castrated rats (48%). All the changes produced in the anterior pituitary gland after castration were prevented by the administration of dihydrotestosterone. The results demonstrate that a stimulation of LH release is followed by an increase of DNA synthesis and cell proliferation of gonadotrophs in the anterior pituitary gland.

  19. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

    Science.gov (United States)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

    2015-01-01

    Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799

  20. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

    Energy Technology Data Exchange (ETDEWEB)

    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. (DNAX Research Inst. of Molecular and Cellular Biology, Palo Alto, CA (United States))

    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.

  1. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

    International Nuclear Information System (INIS)

    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.

    1991-01-01

    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

  2. [Effect of metalaxyl on the synthesis of RNA, DNA and protein in Phytophthora nicotianae].

    Science.gov (United States)

    Wollgiehn, R; Bräutigam, E; Schumann, B; Erge, D

    1984-01-01

    Metalaxyl is used to control diseases caused by fungi of the order of the Perenosporales. We investigated the action of this fungicid eon nucleic acid and protein synthesis in liquid cultures of Phytophthora nicotianae. The uptake of 32P, 3H-uridine, 3H-thymidine and 14C-leucine as precursors of nuclei acid and protein synthesis by the mycelium was not inhibited by metalaxyl. RNA synthesis as indicated by 3H-uridine incorporation was strongly inhibited (about 80%) by 0.5 micrograms/ml of metalaxyl. The inhibition was visible already few minutes after addition of the toxicant. Since the inhibition of incorporation of 3H-thymidine into DNA and of 14C-leucine into protein became significant 2-3 hours later, we conclude that metalaxyl primarily interfers with RNA synthesis. Synthesis of ribosomal RNA is more affected (more than 90%) than that of tRNA (about 55%) and poly(A)-containing RNA. Since in the presence of actinomycin, in contrast to metalaxyl, protein synthesis is inhibited immediately as a consequence of complete inhibition of RNA synthesis and of the short life-time of mRNA, it is also evident that mRNA synthesis is less strongly inhibited, at least during the early period of metalaxyl action. The molecular mechanism of metalaxyl inhibition of the transcription process remains open. The fungicide did not inhibit the activity of a partially purified RNA polymerase isolated from the fungus. On the other hand, the RNA synthesis (14C-UTP-incorporation) by a cell homogenate and by isolated nuclear fractions was inhibited significantly. Possibilities of the molecular action of metalaxyl are discussed. The RNA synthesis of some plant systems (cell cultures of Lycopersicon peruvianum, isolated nuclei from the same cell cultures, purified RNA polymerase from Spinacia oleracea chloroplasts) was not inhibited by metalaxyl, not even at high concentrations.

  3. Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli

    International Nuclear Information System (INIS)

    Rosen, H.; Orman, J.; Rakita, R.M.; Michel, B.R.; VanDevanter, D.R.

    1990-01-01

    Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [ 3 H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNA-membrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify the E. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate

  4. Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

    International Nuclear Information System (INIS)

    Wang, Tzuchien V.; Smith, K.C.

    1986-01-01

    The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed. (Auth.)

  5. Detecting DNA synthesis of neointimal formation after catheter balloon injury in GK and in Wistar rats: using 5-ethynyl-2'-deoxyuridine

    Directory of Open Access Journals (Sweden)

    Guo Jingsheng

    2012-12-01

    Full Text Available Abstract Background Neointimal formation plays an important role in the pathogenesis of coronary restenosis after percutaneous coronary intervention (PCI, especially in patients with diabetes mellitus. Recently, some studies have shown that 5-ethynyl-2'-deoxyuridine (EdU incorporation can serve as a novel alternative to the 5-bromo-2'-deoxyuridine (BrdU antibody detection method for detection of DNA synthesis in regenerating avian cochlea, chick embryo and the adult nervous system. However, few studies have been performed to assess the suitability of EdU for detecting DNA synthesis in vascular neointima. Methods The carotid artery balloon injury model was established in Goto-Kakizaki (GK and Wistar rats. A Cell-LightTM EdU Kit was used to detect EdU-labeled cell nuclei of common carotid arteries at day 7 after catheter balloon injury. Different methods of injecting EdU were tested. The protein levels of proliferating cell nuclear antigen (PCNA and p-Akt (Ser473, as well as the mRNA levels of PCNA were evaluated by Western blotting and quantitative real-time PCR (qRT-PCR, respectively. Immunohistochemical staining was also employed to visualize PCNA-positive cells. Results At day 7 after catheter balloon injury, far more EdU-positive and PCNA-positive cells were observed in GK rats. When comparing groups that received different EdU doses, it was found that the percentage of EdU-positive cells at a dose of 100 mg/kg body weight was than at doses of 25 mg/kg and 50 mg/kg. The number of positive cells was significantly higher in the repeated injection group compared to the single injection group. Further, after balloon injury DNA synthesis in GK rats was more notable than in Wistar rats. Neointimal formation in GK rats was more obvious than in Wistar rats. The protein levels of PCNA and p-Akt (Ser473 and the mRNA levels of PCNA were increased in injured rats as compared to uninjured rats, and were significantly higher in GK rats than in Wistar rats

  6. Synthesis of CdS nanoparticles based on DNA network templates

    International Nuclear Information System (INIS)

    Yao Yong; Song Yonghai; Wang Li

    2008-01-01

    CdS nanoparticles have been successfully synthesized by using DNA networks as templates. The synthesis was carried out by first dropping a mixture of cadmium acetate and DNA on a mica surface for the formation of the DNA network template and then transferring the sample into a heated thiourea solution. The Cd 2+ reacted with thiourea at high temperature and formed CdS nanoparticles on the DNA network template. UV-vis spectroscopy, photoluminescence, x-ray diffraction and atomic force microscopy (AFM) were used to characterize the CdS nanoparticles in detail. AFM results showed that the resulted CdS nanoparticles were directly aligned on the DNA network templates and that the synthesis and assembly of CdS nanoparticles was realized in one step. CdS nanoparticles fabricated with this method were smaller than those directly synthesized in a thiourea solution and were uniformly aligned on the DNA networks. By adjusting the density of the DNA networks and the concentration of Cd 2+ , the size and density of the CdS nanoparticles could be effectively controlled and CdS nanoparticles could grow along the DNA chains into nanowires. The possible growth mechanism has also been discussed in detail

  7. Cell cycle regulation of DNA polymerase beta in rotenone-based Parkinson's disease models.

    Directory of Open Access Journals (Sweden)

    Hongcai Wang

    Full Text Available In Parkinson's disease (PD, neuronal cells undergo mitotic catastrophe and endoreduplication prior to cell death; however, the regulatory mechanisms remain to be defined. In this study, we investigated cell cycle regulation of DNA polymerase β (poly β in rotenone-based dopaminergic cellular and animal models. Incubation with a low concentration (0.25 µM of rotenone for 1.5 to 7 days resulted in a flattened cell body and decreased DNA replication during S phase, whereas a high concentration (2 µM of rotenone exposure resulted in enlarged, multi-nucleated cells and converted the mitotic cycle into endoreduplication. Consistently, DNA poly β, which is mainly involved in DNA repair synthesis, was upregulated to a high level following exposure to 2 µM rotenone. The abrogation of DNA poly β by siRNA transfection or dideoxycytidine (DDC treatment attenuated the rotenone-induced endoreduplication. The cell cycle was reactivated in cyclin D-expressing dopaminergic neurons from the substantia nigra (SN of rats following stereotactic (ST infusion of rotenone. Increased DNA poly β expression was observed in the substantia nigra pars compacta (SNc and the substantia nigra pars reticulate (SNr of rotenone-treated rats. Collectively, in the in vitro model of rotenone-induced mitotic catastrophe, the overexpression of DNA poly β promotes endoreduplication; in the in vivo model, the upregulation of DNA poly β and cell cycle reentry were also observed in the adult rat substantia nigra. Therefore, the cell cycle regulation of DNA poly β may be involved in the pathological processes of PD, which results in the induction of endoreduplication.

  8. A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.

    Science.gov (United States)

    Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

    2010-06-17

    Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity. Copyright 2010 Elsevier B.V. All rights reserved.

  9. DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles.

    Science.gov (United States)

    Du, Huang-Chi; Huang, Hongbing

    2017-10-18

    DNA-encoded chemical libraries have emerged as a cost-effective alternative to high-throughput screening (HTS) for hit identification in drug discovery. A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines. The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The reaction is performed in aqueous solution and does not compromise DNA structural integrity. The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.

  10. Repair of Clustered Damage and DNA Polymerase Iota.

    Science.gov (United States)

    Belousova, E A; Lavrik, O I

    2015-08-01

    Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

  11. Identification of proteins whose synthesis in Saccharomyces cerevisiae is induced by DNA damage and heat shock

    International Nuclear Information System (INIS)

    Gailit, James

    1990-01-01

    Protein synthesis in Saccharomyces cerevisiae after exposure to ultraviolet light (UV) was examined by two-dimensional gel electrophoresis of pulse-labelled proteins. The synthesis of 12 distinct proteins was induced by treatment with UV doses of 10-200 J/m 2 . The induced proteins differed in minimum dose necessary for induction, maximum dose at which induction still occurred and constitutive level present in unirradiated cells. A chemical mutagen, 4-nitroquinoline-1-oxide, induced synthesis of the same proteins. Induction after UV treatment was observed in seven different yeast strains, including three mutants deficient in DNA repair. Synthesis of five of the proteins was also induced by brief heat shock treatment. These five may be members of a family of proteins whose synthesis is regulated by two different pathways responding to different types of stress. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

    Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

    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.

  13. Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

    Science.gov (United States)

    Pasion, S G; Brown, G W; Brown, L M; Ray, D S

    1994-12-01

    In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

  14. Effects of anticonvulsant drugs on the synthesis of DNA and protein by human bone marrow cells in vitro

    International Nuclear Information System (INIS)

    Wickramasinghe, S.N.; Saunders, J.; Williams, G.

    1976-01-01

    Suspensions of human bone marrow cells were incubated with various concentrations of phenobarbitone or phenytoin sodium for 2 h, and the effects of this incubation on the subsequent incorporation of 3 H-thymidine and 3 H-leucine into DNA and protein, respectively, were studied. Both drugs caused a depression of 3 H-thymidine incorporation and this phenomenon was not prevented by the addition of 100 μg of pteroylglutamic acid, folinic acid or 5-methyltetrahydrofolate per ml of marrow culture. The lowest concentration of drug which caused a statistically significant depression of 3 H-thymidine incorporation was 200μg per ml for phenobarbitone and 50 μg per ml for phenytoin sodium. Both phenobarbitone and phenytoin sodium also caused an increase in the incorporation of 3 H-leucine at concentrations of 50 and 20 μg per ml., respectively, suggesting the possibility that a stimulation of protein synthesis within erythropoietic cells may play an important role in the development of anticonvulsant-induced macrocytosis. (authod)

  15. Semiconservative and unscheduled DNA-synthesis of rat thymocytes under the influence of some radioprotecting and radiosensitizing agents

    Energy Technology Data Exchange (ETDEWEB)

    Tempel, K.; Wulffius-Kock, M.; Winkle, J.; Schmerold, I.

    1982-02-01

    The effects of aminoethylisothiuroniumbromide (AET), cysteamine (CY-A), cysteine (CY-E), glutathione (GLU), mercaptoethanol (MA), mercaptopropionylglycine (MPG), N-ethylmaleimide (NEM), metronidazole (MNA), nitroacetophenone (NAP), nitrofurazone (NFA), arabinofuranosylcytosine (araC), fluorouracil (FU), adriamycin (AM), ethidiumbromide (E), bleomycin (BM), and diethyldithiocarbamate (DDC) on the semiconservative and unscheduled incorporation of /sup 3/H-thymidine into the DNA were tested on rat thymocytes in vitro. DNA damage has been measured using the hydroxylapatite system. Unscheduled DNA synthesis was induced by UV-light and/or X-irradiation. The semiconservative DNA synthesis was inhibited by the above substances-with exception of MA and MPG. Aminothioles, NAP, NFA, and BM enhanced, araC, FU, AM, E, and DDC diminished unscheduled DNA synthesis. After alkaline unwinding, the duplex form of DNA decreased under the influence of CY-A, CY-E, GLU, MPG, NEM, NAP, NFA, araC, FU, AM, E, and BM. It is suggested that stimulation of unscheduled DNA synthesis combined with a transient decrease of semiconservative DNA synthesis will amplify the DNA repair capacity of thymocytes, whereas radiation damage may be intensified by araC, FU, AM,E, and DDC - at least partly, through inhibition of unscheduled DNA synthesis. With respect to the action of NAP, NFA, and BM, DNA repair may be concerned in a more indirect manner.

  16. Semiconservative and unscheduled DNA-synthesis of rat thymocytes under the influence of some radioprotecting and radiosensitizing agents

    International Nuclear Information System (INIS)

    Tempel, K.; Wulffius-Kock, M.; Winkle, J.; Schmerold, I.

    1982-01-01

    The effects of aminoethylisothiuroniumbromide (AET), cysteamine (CY-A), cysteine (CY-E), glutathione (GLU), mercaptoethanol (MA), mercaptopropionylglycine (MPG), N-ethylmaleimide (NEM), metronidazole (MNA), nitroacetophenone (NAP), nitrofurazone (NFA), arabinofuranosylcytosine (araC), fluorouracil (FU), adriamycin (AM), ethidiumbromide (E), bleomycin (BM), and diethyldithiocarbamate (DDC) on the semiconservative and unscheduled incorporation of 3 H-thymidine into the DNA were tested on rat thymocytes in vitro. DNA damage has been measured using the hydroxylapatite system. Unscheduled DNA synthesis was induced by UV-light and/or X-irradiation. The semiconservative DNA synthesis was inhibited by the above subtrances-with exception of MA and MPG. Aminothioles, NAP, NFA, and BM enhanced, araC, FU, AM, E, and DDC diminished unscheduled DNA synthesis. After alkaline unwinding, the duplex form of DNA decreased under the influence of CY-A, CY-E, GLU, MPG, NEM, NAP, NFA, araC, FU, AM, E, and BM. It is suggested that stimulation of unscheduled DNA synthesis combined with a transient decrease of semiconservative DNA synthesis will amplify the DNA repair capacity of thymocytes, whereas radiation damage may be intensified by araC, FU, AM,E, and DDC - at least partly, through inhibition of unscheduled DNA synthesis. With respect to the action of NAP, NFA, and BM, DNA repair may be concerned in a more indirect manner. (orig.) [de

  17. Silencing of PINK1 expression affects mitochondrial DNA and oxidative phosphorylation in dopaminergic cells.

    Directory of Open Access Journals (Sweden)

    Matthew E Gegg

    Full Text Available Mitochondrial dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD. Impairment of the mitochondrial electron transport chain (ETC and an increased frequency in deletions of mitochondrial DNA (mtDNA, which encodes some of the subunits of the ETC, have been reported in the substantia nigra of PD brains. The identification of mutations in the PINK1 gene, which cause an autosomal recessive form of PD, has supported mitochondrial involvement in PD. The PINK1 protein is a serine/threonine kinase localized in mitochondria and the cytosol. Its precise function is unknown, but it is involved in neuroprotection against a variety of stress signalling pathways.In this report we have investigated the effect of silencing PINK1 expression in human dopaminergic SH-SY5Y cells by siRNA on mtDNA synthesis and ETC function. Loss of PINK1 expression resulted in a decrease in mtDNA levels and mtDNA synthesis. We also report a concomitant loss of mitochondrial membrane potential and decreased mitochondrial ATP synthesis, with the activity of complex IV of the ETC most affected. This mitochondrial dysfunction resulted in increased markers of oxidative stress under basal conditions and increased cell death following treatment with the free radical generator paraquat.This report highlights a novel function of PINK1 in mitochondrial biogenesis and a role in maintaining mitochondrial ETC activity. Dysfunction of both has been implicated in sporadic forms of PD suggesting that these may be key pathways in the development of the disease.

  18. Inhibition of human arterial smooth muscle (HASM) cell proliferation and collagen synthesis by protamine

    International Nuclear Information System (INIS)

    Drucker, D.E.; Graham, M.F.; Diegelmann, R.F.; Greenfield, L.J.

    1986-01-01

    Atherosclerotic plaques result from vascular smooth muscle cell proliferation and collagen deposition. The authors have been studying factors which modulate HASM cell proliferation and collagen synthesis. HASM cells were isolated from the media of normal human thoracic and infrarenal aortas and grown in vitro. Cell numbers were determined by direct counting and collagen synthesis was measured by incorporation of 3 H-proline into collagenase-digestible protein. In this study, protamine (200 μg/ml) was tested and found to cause a 55% reduction of HASM cell proliferation which was reversible when the cells were returned to control medium or when heparin (100 μg/ml) was added with protamine. Protamine caused a constant 33% decrease in non-collagen protein (NCP) synthesis per cell. In contrast, collagen synthesis was inhibited in dose dependent fashion (88% reduction at 200 μg/ml). Protamine blocks HASM cell proliferation and specifically inhibits collagen production. The exact mechanism of this inhibition is unclear but may be related to a transcriptional event since protamine has a high affinity for DNA

  19. A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

    Directory of Open Access Journals (Sweden)

    Elgjo Kjell

    2009-07-01

    Full Text Available Abstract Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

  20. Dissociation between insulin secretion and DNA synthesis in cultured pancreatic islets

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1985-01-01

    -Tdr incorporation. However, long-term exposure to IBMX did not result in increased DNA content of the islets. Inhibition of the DNA synthesis by 5 mM hydroxyurea resulted in a marked reduction in DNA content of the islets but no decrease in either insulin release or insulin content when expressed per ng DNA...

  1. Dependence of the rate of DNA synthesis in x-irradiated HeLa S3 cells on dose and time after exposure

    International Nuclear Information System (INIS)

    Tolmach, L.J.; Jones, R.W.

    1977-01-01

    After irradiation of randomly dividing cultures of HeLa S3 cells with 220-kV x rays, the rate of DNA synthesis, measured by pulsed incorporation of labeled thymidine, falls nearly exponentially with time (t/sub 1/2/ approximately 1.3 hr), in a dose-independent fashion. The fall is less rapid than that observed after addition of inhibitors of protein synthesis. With doses up to 8 krad, the rate reaches a minimum and begins to increase after 1-3 hr, the minima occurring at lower values and at slightly later times with increasing dose. The increase appears to be roughly linear for about 6 hr, with the slope an inverse function of dose in the range 1-8 krad. About 7-9 hr after the completion of irradiation, the rate again falls, although no more than 10 percent of the cells die sooner than 14 hr after irradiation with 8 krad (and later with smaller doses). Fluorodeoxyuridine-mediated delay in expression of the depression, described previously for doses up to 1 krad, occurs also at higher doses. During the period when the rate per culture rises, the rate in the individual cells, measured autoradiographically, appears to increase also, i.e., the rise presumably does not merely reflect populational shifts. The initial descending portion of the rate curve can be at least partially separated from the ascending portion by administering the total dose in suitably spaced fractions. If interpreted in terms of the model that attributes the initial depression in rate of synthesis to a temporary absence of replicon initiation, the results indicate that initiation is halted by an x-ray dose smaller than 1 krad; that it begins again after a dose-dependent delay amounting to about 0.7 hr after 1 krad and 1.5 hr after 7 krad; and that once begun, the rate of synthesis increases in a dose-dependent fashion. The second depression might derive from synchronization and/or from the imminence of cell death

  2. The proofreading 3'→5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

    International Nuclear Information System (INIS)

    Khare, Vineeta; Eckert, Kristin A.

    2002-01-01

    The 3'→5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase γ (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'→5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity

  3. DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, L F; Glaubiger, D L [National Cancer Inst., Bethesda, MD (USA). Pediatric Oncology Branch; Kraemer, K H; Waters, H L [National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Carcinogenesis; Kohn, K W [National Cancer Inst., Bethesda, MD (USA). Lab. of Molecular Pharmacology

    1981-01-01

    8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultraviolet radiation (UV-A) inhibits DNA synthesis in lymphocytes in vitro and in vivo. 8-MOP binds reversibly to DNA in the dark; when exposed to UV-A, covalent monoadducts and cross-links are formed with the DNA. The present study correlates the cytotoxic effects of 8-MOP plus UV-A with DNA crosslinking. E-B virus transformed human lymphoblastoid cells were suspended in a colorless salt solution containing 8-MOP and exposed to UV-A from fluorescent lamps filtered to remove radiation below 320 nm (22.5 J/m/sup 2/-sec). Cells were then returned to complete medium and assayed for survival (by daily counts of viable cells and by cloning in microtiter wells) and for DNA crosslinking by alkaline elution. 8-MOP alone or UV-A alone resulted in minimal to no alterations in survivial or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01-1.0 ..mu..g/ml) for 3 different UV-A doses (3000-15000 J/m/sup 2/). The surviving fraction declined exponentially as a function of the relative number of DNA crosslinks.

  4. The nature of newly-synthesized DNA in irradiated lymphoid cells, measured by a technique sensitive to changes after low doses

    International Nuclear Information System (INIS)

    Olsen, I.; Herbert, L.; Harris, G.; Cramp, W.A.; Hesslewood, I.P.; Parker, J.

    1978-01-01

    We have investigated the post-irradiation synthesis of DNA in a lymphoid cell line (LDV) obtained from normal human peripheral blood and maintained in culture. For doses up to Gy (1 kilorad) the repair of DNA damage in these cells was rapid and complete. However, when DNA strand elongation was assayed in apparently fully repaired cells the new DNA was grossly abnormal. Hydroxapathie chromatography was used to examine lesions in prelabelled DNA as well as strand elongation. Because of the sensitivity of this technique we have been able to show that the repair process is error prone. (orig.) [de

  5. Effects of radiation on DNA

    International Nuclear Information System (INIS)

    Medina, V.F.O.

    1978-01-01

    Irradiation has been shown to depress DNA (deoxyribonucleic acid) synthesis resulting in deficient DNA synthesis. In one experiment, Hela S 3 cells completed the next division after a dose of 500 rads to 200 kw X-rays. Another experiment showed that the amount of DNA synthesized was dependent on the stage in the generation cycle at which the cells are irradiated (Giffites and Tolmach, 1975). DNA synthesis was measured by radioactive thymidine incorporation. The smallest deficiency (20-35%) after a dose of 500 rad X-ray was observed in Hela S 3 cells irradiated in early G 1 or early G 2 , while the greatest deficiency (55-70*) after 500 rad X-ray was found in cells irradiated at mitosis or at the Gsub(1)/S transition. Using velocity sedimentation in alkaline gradients of the DNA from hamster, Elkind, et al 1972, studied repair processes as a function of X-ray dose. DNA containing material released by alkaline lysis was found initially contained in a complex-containing lipid, the sedimentation of which was anomalous relative to denatured RNA from unirradated cells. Doses of X-rays small enough to be in the range that permits high survival (100-800 rads) speed the resolution of single-stranded DNA from this DNA complex, giving rise to a species having a number average molecular weight of 2 x 10 8 daltons. Larger doses greater than 1000 to 2000 rads resulted in a degradation of these DNA strands. Incubation after irradiation resulted in the rapid repair of damage, although the rate of repair of damage to the complex resulted in a reassociation of lipid and DNA. This evidence supports the possibility that a large DNA-membrane structure is a principal target of radiation

  6. Attenuated Shigella as a DNA Delivery Vehicle for DNA-Mediated Immunization

    Science.gov (United States)

    Sizemore, Donata R.; Branstrom, Arthur A.; Sadoff, Jerald C.

    1995-10-01

    Direct inoculation of DNA, in the form of purified bacterial plasmids that are unable to replicate in mammalian cells but are able to direct cell synthesis of foreign proteins, is being explored as an approach to vaccine development. Here, a highly attenuated Shigella vector invaded mammalian cells and delivered such plasmids into the cytoplasm of cells, and subsequent production of functional foreign protein was measured. Because this Shigella vector was designed to deliver DNA to colonic mucosa, the method is a potential basis for oral and other mucosal DNA immunization and gene therapy strategies.

  7. DNA synthesis in the pituitary gland of the rat: effect of sulpiride and clomiphene.

    Science.gov (United States)

    Burdman, J A; Szijan, I; Jahn, G A; Machiavelli, G; Kalbermann, L E

    1979-09-15

    Sulpiride administration to rats releases prolactin and increases DNA replication in the anterior pituitary gland. Clomiphene prevents the stimulation of DNA synthesis produced by sulpiride, but does not affect prolactin release from the gland. These findings suggest that the intracellular prolactin content of the anterior pituitary gland plays a role in the regulation of DNA synthesis through a mechanism mediated by oestrogens.

  8. Age-related variation in the DNA-repair synthesis after UV-C irradiation in unstimulated lymphocytes of healthy blood donors

    International Nuclear Information System (INIS)

    Kovacs, E.; Weber, W.; Mueller, H.

    1984-01-01

    UV-C light-induced DNA-repair synthesis was studied in unstimulated lymphocytes of 51 healthy blood donors aged between 17 and 74 years. The evaluation included (1) the spontaneous DNA-synthesis in unirradiated lymphocytes with and without hydroxyurea, (2) the DNA-repair synthesis in lymphocytes irradiated with UV-light. The interindividual variation was significantly higher than the methodological variation ascertained in 24 persons in whom 2 determinations were carried out. In blood donors aged between 17 and 39 years, the spontaneous DNA synthesis, both with and without hydroxyurea, was significantly lower than in older individuals. The DNA-repair synthesis was dependent on the dose of UV-C light between 2 and 16 J/m 2 . There were no significant differences in DNA-repair synthesis in the age range 17-74 years. The variations in rate of DNA-repair synthesis were wider in older (44-74 years), than in younger individuals. (orig.)

  9. A new human photosensitive subject with a defect in the recovery of DNA synthesis after ultraviolet-light irradiation

    International Nuclear Information System (INIS)

    Fujiwara, Y.; Ichihashi, M.; Kano, Y.; Goto, K.; Shimizu, K.

    1981-01-01

    A non-sensitive, 8-yr-old male patient (termed UV81KO) with only acute recurrent sunburns and without any other physical or neuromental retardations was studied. The patient's skin exhibited lowered minimal erythema doses between 280 and 300 nm monochromatic wavelengths without delayed peaking of erythema. UV81KO skin fibroblasts in culture was 5-fold more sensitive to 254 nm UV killing than normal cells, though the response of obligatory heterozygotes was normal. UV81KO cells were also more sensitive to killings by fluorescent sunlamp (295-300 nm UV-B) radiation, 4-nitroquinoline-1-oxide, and N-hydroxy-acetyl aminofluorene, but not by monofunctional decarbamoyl mitomycin C, bifunctional mitomycin C, and alkylating agents (methyl methanesulfonate, ethyl methanesulfonate, N-methyl-N-nitrosourea). Assays for unscheduled DNA synthesis, T4 endonuclease V-susceptible sites (pyrimidine dimers), endogenous excision-break accumulation by arabinofuranosyl cytosine-plus-hydroxyurea, single-strand-break rejoining, and molecular-weight increase of pulse-chased DNA in irradiated cells indicated no apparently detectable defects in nucleotide-excision repair processes and in replicative bypass in UV81KO cells. Despite the repair proficiency as such, UV81KO cells showed the defective recovery of DNA synthesis after 254 nm UV irradiation with 1 and 5 J/m2, at which dose the recovery occurred in normal cells. The base line level of sister-chromatid exchanges (SCEs) was higher in UV81KO cells (10-12 SCEs/cell) than in normal cells (5 SCEs/cell), although the induction rate of SCEs by 254 nm UV in UV81KO cells was the same as in normal cells. Such clinical, cellular and molecular characteristics and comparison to those in the other photodermatoses (xeroderma pigmentosum, Cockayne's syndrome, the 11961 disorder, Bloom's syndrome) can make a clear distinction of UV81KO from the others

  10. A new human photosensitive subject with a defect in the recovery of DNA synthesis after ultraviolet-light irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Y.; Ichihashi, M.; Kano, Y.; Goto, K.; Shimizu, K.

    1981-09-01

    A non-sensitive, 8-yr-old male patient (termed UV81KO) with only acute recurrent sunburns and without any other physical or neuromental retardations was studied. The patient's skin exhibited lowered minimal erythema doses between 280 and 300 nm monochromatic wavelengths without delayed peaking of erythema. UV81KO skin fibroblasts in culture was 5-fold more sensitive to 254 nm UV killing than normal cells, though the response of obligatory heterozygotes was normal. UV81KO cells were also more sensitive to killings by fluorescent sunlamp (295-300 nm UV-B) radiation, 4-nitroquinoline-1-oxide, and N-hydroxy-acetyl aminofluorene, but not by monofunctional decarbamoyl mitomycin C, bifunctional mitomycin C, and alkylating agents (methyl methanesulfonate, ethyl methanesulfonate, N-methyl-N-nitrosourea). Assays for unscheduled DNA synthesis, T4 endonuclease V-susceptible sites (pyrimidine dimers), endogenous excision-break accumulation by arabinofuranosyl cytosine-plus-hydroxyurea, single-strand-break rejoining, and molecular-weight increase of pulse-chased DNA in irradiated cells indicated no apparently detectable defects in nucleotide-excision repair processes and in replicative bypass in UV81KO cells. Despite the repair proficiency as such, UV81KO cells showed the defective recovery of DNA synthesis after 254 nm UV irradiation with 1 and 5 J/m2, at which dose the recovery occurred in normal cells. The base line level of sister-chromatid exchanges (SCEs) was higher in UV81KO cells (10-12 SCEs/cell) than in normal cells (5 SCEs/cell), although the induction rate of SCEs by 254 nm UV in UV81KO cells was the same as in normal cells. Such clinical, cellular and molecular characteristics and comparison to those in the other photodermatoses (xeroderma pigmentosum, Cockayne's syndrome, the 11961 disorder, Bloom's syndrome) can make a clear distinction of UV81KO from the others.

  11. Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells

    International Nuclear Information System (INIS)

    Barra, R.; Beres, B.; Koch, M.R.; Lea, M.A.

    1976-01-01

    The effects of exogenous proteins on the incorporation of [ 3 H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A 2 , 5123C and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10 to 20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100 to 400μg/ml. In experiments with lower cell concentrations, a 60% or greater increase in [ 3 H]-thymidine incorporation could be obtained with total calf thymus histone and with Fl and arginine-rich histones from rat liver. At concentrations of 1 to 2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80 to 90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10μg/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis. (author)

  12. Differences in the regulation by poly(ADP-ribose) of repair of DNA damage from alkylating agents and ultraviolet light according to cell type

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.; Bodell, W.J.; Morgan, W.F.; Zelle, B.

    1983-08-10

    Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cells influenced the responses to DNA damage from methyl methanesulfonate, but not from ultraviolet light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but only stimulated repair replication in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects may be due to disturbance of precursor pathways and irrelevant to the role of poly(ADP-ribose) in repair. Previous claims that 3-aminobenzamide stimulates repair synthesis after exposure to UV light are probably artifacts, because the stimulations are only observed in lymphocytes in the presence of a high concentration of hydroxyurea that itself inhibits repair. The initial inhibition of semiconservative DNA synthesis and the excision of the major alkylation products and pyrimidine dimers were unaffected by 3-aminobenzamide. In general poly(ADP-ribose) synthesis appears to be uniquely involved in regulating the ligation stage of repair of alkylation damage but not ultraviolet damage. By regulating the ligation efficiency, poly(ADP-ribosylation) modulates the dynamic balance between incision and ligation, so as to minimize the frequency of DNA breaks. The ligation stage of repair of UV damage appears different and is not regulated by poly(ADP-ribosylation).

  13. Direct on-chip DNA synthesis using electrochemically modified gold electrodes as solid support

    Science.gov (United States)

    Levrie, Karen; Jans, Karolien; Schepers, Guy; Vos, Rita; Van Dorpe, Pol; Lagae, Liesbet; Van Hoof, Chris; Van Aerschot, Arthur; Stakenborg, Tim

    2018-04-01

    DNA microarrays have propelled important advancements in the field of genomic research by enabling the monitoring of thousands of genes in parallel. The throughput can be increased even further by scaling down the microarray feature size. In this respect, microelectronics-based DNA arrays are promising as they can leverage semiconductor processing techniques with lithographic resolutions. We propose a method that enables the use of metal electrodes for de novo DNA synthesis without the need for an insulating support. By electrochemically functionalizing gold electrodes, these electrodes can act as solid support for phosphoramidite-based synthesis. The proposed method relies on the electrochemical reduction of diazonium salts, enabling site-specific incorporation of hydroxyl groups onto the metal electrodes. An automated DNA synthesizer was used to couple phosphoramidite moieties directly onto the OH-modified electrodes to obtain the desired oligonucleotide sequence. Characterization was done via cyclic voltammetry and fluorescence microscopy. Our results present a valuable proof-of-concept for the integration of solid-phase DNA synthesis with microelectronics.

  14. Defective recovery of semi-conservative DNA synthesis in xeroderma pigmentosum cells following split-dose ultraviolet irradiation

    International Nuclear Information System (INIS)

    Moustacchi, E.; Ehmann, U.K.; Friedberg, E.C.

    1979-01-01

    In normal human fibroblasts the authors observe an enhancement of the recovery of the rate of semi-conservative DNA synthesis after split-dose UV-irradation relative to a single total UV dose. The enhanced recovery is totally absent in both a xeroderma pigmentosum variant line and two xeroderma pigmentosum lines belonging to complementation groups A and C. (Auth.)

  15. Effect of irradiation on unscheduled DNA synthesis induced by 4-nitroquinoline in tracheal epithelium of rats

    International Nuclear Information System (INIS)

    Hahn, F.F.; Kennedy, R.; Brooks, A.L.

    1986-01-01

    Unscheduled DNA synthesis (UDS) was determined in rat epithelium by autoradiographic techniques to determine the influence of prior irradiation on the ability of the cells to repair mutagenic damage induced by 4-nitroquionoline (4NQO). UDS was stimulated by in vitro exposure to 4NPO. However, prior whole-body irradiation of rats with either 50 or 300 rad did not alter the UDS induced by 4NQO. The results of this study do not support the hypothesis that irradiation can induce DNA repair enzymes in respiratory tract epithelium. 5 references, 3 figures

  16. DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

    1988-08-01

    The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after ..gamma..-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation.

  17. DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

    International Nuclear Information System (INIS)

    Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

    1988-01-01

    The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after γ-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation. (author)

  18. The studies of effects of 60Co γ-rays on DNA damage and repair in tumor cells

    International Nuclear Information System (INIS)

    Su Liaoyuan; Huang Hanxian; Cen Jiannong

    1997-06-01

    The effects of 60 Co γ-rays and its combination with hyperthermia on DNA strand breaks and their repair in L 5178y cells were studied using alkaline elution technique. When the cells were heated at 43 degree C for 30 min, there was obvious inhibition of repair of DNA damage caused by γ-irradiation. The hyperthermia before irradiation produced better inhibiting effect than that after irradiation. The effects of radiation on DNA strand breaks and its repair in HL-60 cells and HL-60 (VCR) cells were analyzed using technique of hydroxyapatite chromatography and the results sowed that the extent of DNA strand breaks in HL-60 cells and HL-60 (VCR) cells induced by irradiation was not markedly different, but the power of repair of strand breaks and the radioresistance of function of DNA synthesis in HL-60 (VCR) cells were higher than those in HL-60 cells. The difference was obvious. The results suggest that the heterogeneity of radiosensitivity of leukemia cells is correlated with its drug resistance. (10 refs., 3 tabs.)

  19. Induction of DNA synthesis and apoptosis are separable functions of E2F-1

    DEFF Research Database (Denmark)

    Phillips, A C; Bates, S; Ryan, K M

    1997-01-01

    The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic...... response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant...... apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E...

  20. Decreased cell survival and DNA repair capacity after UVC irradiation in association with down-regulation of GRP78/BiP in human RSa cells

    International Nuclear Information System (INIS)

    Zhai Ling; Kita, Kazuko; Wano, Chieko; Wu Yuping; Sugaya, Shigeru; Suzuki, Nobuo

    2005-01-01

    In contrast to extensive studies on the roles of molecular chaperones, such as heat shock proteins, there are only a few reports about the roles of GRP78/BiP, an endoplasmic reticulum (ER) stress-induced molecular chaperone, in mammalian cell responses to DNA-damaging stresses. To investigate whether GRP78/BiP is involved in resistance to a DNA-damaging agent, UVC (principally 254 nm in wavelength), we established human cells with down-regulation of GRP78/BiP by transfection of human RSa cells with antisense cDNA for GRP78/BiP. We found that the transfected cells showed higher sensitivity to UVC-induced cell death than control cells transfected with the vector alone. In the antisense-cDNA transfected cells, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4) photoproducts) in vivo and DNA synthesis activity of whole cell extracts to repair UVC-irradiated plasmids in vitro were remarkably decreased compared with those in the control cells. Furthermore, the antisense-cDNA transfected cells also showed slightly higher sensitivity to cisplatin-induced cell death than the control cells. Cisplatin-induced DNA damage is primarily repaired by nucleotide excision repair, like UVC-induced DNA damage. The present results suggest that GRP78/BiP plays a protective role against UVC-induced cell death possibly via nucleotide excision repair, at least in the human RSa cells tested

  1. Poly(ADP-ribose) synthesis following DNA damage in cells heterozygous or homozygous for the xeroderma pigmentosum genotype

    International Nuclear Information System (INIS)

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Treatment of normal human cells with DNA-damaging agents such as uv light or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) stimulates the conversion of NAD to the chromosomal polymer poly(ADP-ribose) which in turn results in a rapid depletion of the cellular NAD pool. The effect of uv light or MNNG on the NAD pools of seven cell lines of human fibroblasts either homozygous or heterozygous for the xeroderma pigmentosum genotype has been studied. Xeroderma pigmentosum cells of genetic complementation groups A, C, and D are deficient in the excision repair of DNA damage caused by uv light. Following uv treatment, the NAD content of these cells was unchanged or only slightly reduced. All of the cell lines are able to excise DNA damage caused by MNNG and all of the cell lines had a greatly reduced content of NAD following MNNG treatment. The results demonstrate a close relationship between the conversion of NAD to poly(ADP-ribose) and DNA excision repair in human cells

  2. Saturation of DNA repair in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Ahmed, F E; Setlow, R B

    1979-01-01

    Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20 J/m/sup 2/. We measured excision repair in normal human fibroblasts up to 80 J/m/sup 2/. The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus, and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24h following two 20 J/m/sup 2/ doses separated by 24h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the rate limiting steps in repair.

  3. Semi-conservative deoxyribonucleic acid synthesis in unirradiated and ultraviolet-irradiated xeroderma pigmentosum and normal human skin fibroblasts

    International Nuclear Information System (INIS)

    Rude, J.M.; Friedberg, E.C.

    1977-01-01

    Rates of semiconservative DNA synthesis have been investigated in asynchronous xeroderma pigmentosum (XP), XP variant, and normal human skin fibroblasts using the technique of cellular autoradiography. In unirradiated cells, no differences in DNA synthesis rates were detected among the three cell strains. Exposure to UV radiation caused the rate of DNA synthesis to decrease for at least three hours in all three cell strains. In the normal cell strain, recovery of the DNA synthetic rate occurred at later times following a UV fluence of 5 J/m 2 . At this same UV fluence, recovery was absent in classical XP cells during a 24 h post-irradiation period while it was slower than normal in XP variant cells. When the UV fluence to classical XP and XP variant cells was reduced so that survival in all three cell strains was approximately the same (25%), recovery of the DNA synthetic rate was similar in all three cell strains. These results are discussed in terms of current models of DNA replication in UV-irradiated cells and indicate: (1) that pyrimidine dimers are very effective blocks to DNA synthesis and (2) that there is no inherent defect in semiconservative DNA synthesis in either classical XP or XP variant cells which is independent of a defect in DNA repair capacity

  4. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  5. Changes in the synthesis of DNA, RNA and protein during somatic embryogenesis in wheat (triticum aestivum L.)

    International Nuclear Information System (INIS)

    Cui Kairong; Wang Xiaozhe; Chen Xiong; Wang Yafu

    1997-01-01

    Embryogenic and non-embryogenic callus formed from immature embryo of wheat (Triticum aestivum L.) in N 6 B 5 MS medium I supplemented with 2,4-D 2 mg/L, KT 0.5 mg/L, LH300 mg/L, sucrose 3% were sub-cultured and transferred respectively to N 6 B 5 MS medium II (2,4-D was decreased to 0.5 mg/L and 4 mol/L proline was added). Somatic embryos obtained from embryogenic callus, and plantlet formed from non-embryogenic callus through organogenesis respectively. By incorporation of 3 H-thymidine, 3 H-uridine and 3 H-leucine into DNA, RNA and protein respectively, the rate of synthesis of DNA, RNA and protein during somatic embryogenesis were measured. A large amount of RNA and protein synthesized during the early somatic embryogenesis. The activities of RNA and protein synthesis reached the peak on the 4th and the 8th day respectively, then decreased a little, but kept a high level. The synthesis of DNA increased apparently during the early stage. No apparent change occurred when the embryogenic cell masses formed. The synthesis rate of RNA and protein in non-embryogenic callus were much less than that in embryogenic callus. Actinomycin and cycloheximide inhibited not only the synthesis of nucleic acid and protein, but also the growth of embryogenic callus and somatic embryogenesis. The earlier the inhibitors were added, the greater the influence was caused. The results indicate that the active expression of corresponding genes of wheat is the molecular base of somatic embryogenesis

  6. Yield of DNA strand breaks and their relationship to DNA polymerase I-dependent repair synthesis and ligation following x-ray exposure of toluene-treated Escherichia coli

    International Nuclear Information System (INIS)

    Billen, D.

    1981-01-01

    In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is observed. This is an exaggerated repair synthesis which can be abruptly terminated by the addition of the DNA ligase cofactor, nicotinamide adenine dinucleotide. This communication describes experiments which bear on the relationship between measurable strand breaks, DNA polymerase I-directed, exaggerated repair synthesis, and strand-break repair

  7. WR-1065 and radioprotection of vascular endothelial cells. I. Cell proliferation, DNA synthesis and damage

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Kang, H.J.; Baumann, F.E.; Blazek, E.R.

    1996-01-01

    Normal tissue toxicity limits radiation therapy and could depend on the extent of damage to the vascular endothelium. Aminothiols such as WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] provide radioprotection for normal tissues, but little is known about how the aminothiols specifically affect the endothelium. Bovine aortic endothelial cells in culture were exposed to WR-1065 for 2 h before irradiation ( 137 Cs γ rays, 1 Gy/min). Alone, WR-1065 demonstrated an antiproliferative effect that was related to dose (0.5-4 mM) and was evident by lowered counts of adherent cells 48 h after exposure. WR-1065 was clearly radioprotective when assessed by colony formation and incorporation of [ 3 H]thymidine. However, when the number of adherent cells was evaluated, radioprotection appeared to be slight and evident only in logarithmically growing cells. WR-1065 at 2 mM suppressed single-strand DNA breaks after 3 Gy by 22% and double-strand breaks after 9 Gy by 47%. Also in the irradiated cells, WR-1065 more than doubled the rate of progression of cells from G 1 to S phase. WR-1065 pretreatment elevated cellular glutathione (GSH) content more than twofold. Although pretreatment with buthionine sulfoximine inhibited the elevation of GSH, the radioprotective impact of WR-1065 on total DNA strand breaks and colony formation was unaffected. These results suggest that WR-1065 may enable tissue recovery from irradiation by promoting the replication of endothelial cells, possibly by mechanisms independent of GSH. 46 refs., 6 figs., 2 tabs

  8. Neurotoxicity of cytarabine (Ara-C) in dorsal root ganglion neurons originates from impediment of mtDNA synthesis and compromise of mitochondrial function.

    Science.gov (United States)

    Zhuo, Ming; Gorgun, Murat F; Englander, Ella W

    2018-06-01

    Peripheral Nervous System (PNS) neurotoxicity caused by cancer drugs hinders attainment of chemotherapy goals. Due to leakiness of the blood nerve barrier, circulating chemotherapeutic drugs reach PNS neurons and adversely affect their function. Chemotherapeutic drugs are designed to target dividing cancer cells and mechanisms underlying their toxicity in postmitotic neurons remain to be fully clarified. The objective of this work was to elucidate progression of events triggered by antimitotic drugs in postmitotic neurons. For proof of mechanism study, we chose cytarabine (ara-C), an antimetabolite used in treatment of hematological cancers. Ara-C is a cytosine analog that terminates DNA synthesis. To investigate how ara-C affects postmitotic neurons, which replicate mitochondrial but not genomic DNA, we adapted a model of Dorsal Root Ganglion (DRG) neurons. We showed that DNA polymerase γ, which is responsible for mtDNA synthesis, is inhibited by ara-C and that sublethal ara-C exposure of DRG neurons leads to reduction in mtDNA content, ROS generation, oxidative mtDNA damage formation, compromised mitochondrial respiration and diminution of NADPH and GSH stores, as well as, activation of the DNA damage response. Hence, it is plausible that in ara-C exposed DRG neurons, ROS amplified by the high mitochondrial content shifts from physiologic to pathologic levels signaling stress to the nucleus. Combined, the findings suggest that ara-C neurotoxicity in DRG neurons originates in mitochondria and that continuous mtDNA synthesis and reliance on oxidative phosphorylation for energy needs sensitize the highly metabolic neurons to injury by mtDNA synthesis terminating cancer drugs. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Effects of exogenous fatty acids and inhibition of de novo fatty acid synthesis on disaturated phosphatidylcholine production by fetal lung cells and adult type II cells.

    Science.gov (United States)

    Maniscalco, W M; Finkelstein, J N; Parkhurst, A B

    1989-05-01

    De novo fatty acid synthesis may be an important source of saturated fatty acids for fetal lung disaturated phosphatidylcholine (DSPC) production. To investigate the roles of de novo fatty acid synthesis and exogenous fatty acids, we incubated dispersed fetal lung cells and freshly isolated adult type II cells with exogenous palmitate and oleate and measured DSPC synthesis. Unlike adult type II cells, fetal lung cells did not increase DSPC synthesis when exogenous palmitate was available; adult type II cells increased DSPC synthesis by 70% in the presence of palmitate. Exogenous oleate decreased DSPC synthesis by 48% in fetal cells but not in adult type II cells. Incubation of fetal lung cells with TOFA [2-furancarboxylate, 5-(tetradecyloxy)-sodium], a metabolic inhibitor of fatty acid synthesis, decreased fatty acid synthesis by 65%. There was a simultaneous 56% inhibition of DSPC production, but no effect on protein, DNA, or glyceride-glycerol production, measured by precursor incorporation. The inhibition of DSPC synthesis associated with TOFA was partially prevented by exogenous palmitate but not oleate. Fetal cells prepared from explants that had been cultured in dexamethasone also had TOFA-associated inhibition of DSPC synthesis that was similar to non-dexamethasone-exposed cells. These studies suggest that under baseline conditions of low fatty acid availability, such as in the fetus, de novo fatty acid synthesis in fetal cells, but not in adult type II cells, provides sufficient saturated fatty acids to support maximal DSPC production. Inhibition of de novo fatty acid synthesis resulting in decreased DSPC production in fetal lung cells in conditions of low fatty acid availability suggests that fatty acid synthesis may be central to maintain DSPC synthesis in the fetus.

  10. Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

    Science.gov (United States)

    Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

    2013-01-01

    Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

  11. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Yezzi, M.J.

    1985-01-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, was compared to the wild-type cell, CHO-SC1, in single- and split-radiation-dose schemes. When the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal X-ray damage. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Distinct perturbations in the cell-cycle progression were noted following heat alone or heat with radiation. A delay in the progression of synchronized G 1 -phase and S-phase cells was demonstrated autoradiographically after inhibition of protein synthesis. In addition, treated S-phase cells showed a transient increase in the percent labelled cells after the cells were returned to their normal growth temperature of 35 0 C. This observation was suggestive of an unusual pattern of DNA synthesis during the recovery period. Split-dose experiments were done using incubation with cycloheximide to chemically inhibit protein synthesis. Both the chemical and thermal inhibition of protein synthesis substantiate its necessity for the repair of sublethal damage

  12. Correlation between ultraviolet survival and DNA repair efficiency in mouse cell hybrids and their parent lines

    International Nuclear Information System (INIS)

    Limbosch, S.

    1982-01-01

    Three hybrid cell lines formed between mouse lymphoma (LS) and mouse fibroblasts (A9) have been tested for their capacity to perform unscheduled DNA synthesis; their recovery characteristics after uv irradiation have also been studied to determine if DNA repair is implicated in the high survival observed in one hybrid (clone 3). The results of these investigations indicate that hybrid clone 3 was distinguishable from the more uv sensitive parental and other hybrid cell lines by its higher uv-induced unscheduled DNA synthesis, its greater clonogenic survival in plateau phase, and its faster recovery when maintained in conditioned medium after irradiation. The simultaneous increase of these three properties in hybrid clone 3 suggest that, by three different approaches, we have evidenced the same molecular process, a process involved in the elimination of potentially lethal damage, most probably the excision repair pathway. This report also shows that the low efficiency in excision repair in the parent line A9 is probably not due to deletion but rather to repression of the relevant gene(s) and that somatic cell hybridization can result in a stimulation of a previously poorly expressed repair process

  13. Semi-conservative deoxyribonucleic acid synthesis in unirradiated and ultraviolet-irradiated xeroderma pigmentosum and normal human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Rude' e, J.M.; Friedberg, E.C.

    1977-03-01

    Rates of semiconservative DNA synthesis have been investigated in asynchronous xeroderma pigmentosum (XP), XP variant, and normal human skin fibroblasts using the technique of cellular autoradiography. In unirradiated cells, no differences in DNA synthesis rates were detected among the three cell strains. Exposure to uv radiation caused the rate of DNA synthesis to decrease for at least three hours in all three cell strains. In the normal cell strain, recovery of the DNA synthetic rate occurred at later times following a uv fluence of 5 J/m2. At this same uv fluence, recovery was absent in classical XP cells during a 24 h post-irradiation period while it was slower than normal in XP variant cells. When the uv fluence to classical XP and XP variant cells was reduced so that survival in all three cell strains was approximately the same (25%), recovery of the DNA synthetic rate was similar in all three cell strains. These results are discussed in terms of current models of DNA replication in uv-irradiated cells and indicate: (1) that pyrimidine dimers are very effective blocks to DNA synthesis and (2) that there is no inherent defect in semi-conservative DNA synthesis in either classical XP or XP variant cells which is independent of a defect in DNA repair capacity.

  14. DNA synthesis in ataxia telangiectasia

    OpenAIRE

    Jaspers, Nicolaas

    1985-01-01

    textabstractAfter the discovery that cultured cells from AT patients are hypersensitive to ionizing radiation the suggestion was made that AT-could be the 1 X-ray-analogue 1 of xeroderma pigmentosum. The latter syndrome (XP) is characterized by hypersensitivity to short-wave UV-radiation, caused by a reduced ability to properly remove UV-induced DNA damage. The evidence for a DNA repair defect in AT cells is not as strong as in the case of XP (see section 2.2.5 of this thesis). Different XP p...

  15. Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR.

    Science.gov (United States)

    Nomoto, Mika; Tada, Yasuomi

    2018-01-01

    Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  16. An endogenous immune adjuvant released by necrotic cells for enhancement of DNA vaccine potency.

    Science.gov (United States)

    Dorostkar, Rohollah; Bamdad, Taravat; Parsania, Masoud; Pouriayevali, Hassan

    2012-12-01

    Improving vaccine potency in the induction of a strong cell-mediated cytotoxicity can enhance the efficacy of vaccines. Necrotic cells and the supernatant of necrotic tumor cells are attractive adjuvants, on account of their ability to recruit antigen-presenting cells to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells. To evaluate the utility of supernatant of necrotic tumor cells as a DNA vaccine adjuvant in a murine model. The supernatant of EL4 necrotic cells was co-administered with a DNA vaccine expressing the glycoprotein B of Herpes simplex virus-1 as an antigen model under the control of Cytomegalovirus promoter. C57BL/6 mice were vaccinated three times at two weeks intervals with glycoprotein B DNA vaccine and supernatant of necrotic EL4 cells. Five days after the last immunization, cell cytotoxicity, IFN-γ and IL-4 were evaluated. The obtained data showed that the production of IFN-γ from the splenocytes after antigenic stimulation in the presence of the supernatant of necrotic EL4 cells was significantly higher than the other groups (pEL4 cells in the mice immunized with DNA vaccine and supernatant of necrotic EL4 cells comparing to the other groups (p<0.001). The supernatant of necrotic cells contains adjuvant properties that can be considered as a candidate for tumor vaccination.

  17. Relationships among cell survival, O6-alkylguanine-DNA alkyltransferase activity, and reactivation of methylated adenovirus 5 and herpes simplex virus type 1 in human melanoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Maynard, K.; Parsons, P.G.; Cerny, T.; Margison, G.P. (Queensland Institute of Medical Research, Herston (Australia))

    1989-09-01

    O6-Alkylguanine-DNA alkyltransferase (ATase) activity and host cell reactivation (HCR) of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC)-methylated viruses were compared in human melanoma cell lines that were sensitive or resistant to killing by the antitumor DNA-methylating agent MTIC. Enhanced HCR of adenovirus 5 (defined as the Mer+ phenotype) generally showed a semiquantitative correlation with the natural or induced resistance of the host cells to the toxic effects of MTIC and to the level of ATase activity. However, one MTIC-resistant cell line was found (MM170) which had a low level of ATase and intermediate HCR of adenovirus. The HCR of herpes simplex virus type 1 (HSV-1) was enhanced in the Mer+ cells that had natural resistance to MTIC compared with Mer- cells. On the other hand, HCR of HSV-1 in Mer+ cells with induced resistance to MTIC was similar to that in Mer- cells. Neither adenovirus 5 nor HSV-1 infection induced ATase activity in Mer- cells. This indicates that resistance to the toxic effects of methylating agents is not invariably associated with high levels of ATase activity in human melanoma cells. Furthermore, while induction of the Mer+ phenotype from Mer- cells was usually accompanied by the recovery of ATase activity, induced Mer+ cells had less proficient repair than natural Mer+ cells, as judged quantitatively by slightly lower cellular resistance and qualitatively by deficient HCR response for HSV-1. These results suggest that the Mer- and induced Mer+ cells lack an ATase-independent DNA repair mechanism. No differences in MTIC-induced DNA repair synthesis or strand breaks were found between the Mer-, natural Mer+, and induced Mer+ phenotypes. However, UV-induced DNA repair synthesis was higher in the natural Mer+ than in the Mer- or induced Mer+ cells, both of which had increased cellular sensitivity to the antimetabolites methotrexate and hydroxyurea.

  18. Relationships among cell survival, O6-alkylguanine-DNA alkyltransferase activity, and reactivation of methylated adenovirus 5 and herpes simplex virus type 1 in human melanoma cell lines

    International Nuclear Information System (INIS)

    Maynard, K.; Parsons, P.G.; Cerny, T.; Margison, G.P.

    1989-01-01

    O6-Alkylguanine-DNA alkyltransferase (ATase) activity and host cell reactivation (HCR) of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC)-methylated viruses were compared in human melanoma cell lines that were sensitive or resistant to killing by the antitumor DNA-methylating agent MTIC. Enhanced HCR of adenovirus 5 (defined as the Mer+ phenotype) generally showed a semiquantitative correlation with the natural or induced resistance of the host cells to the toxic effects of MTIC and to the level of ATase activity. However, one MTIC-resistant cell line was found (MM170) which had a low level of ATase and intermediate HCR of adenovirus. The HCR of herpes simplex virus type 1 (HSV-1) was enhanced in the Mer+ cells that had natural resistance to MTIC compared with Mer- cells. On the other hand, HCR of HSV-1 in Mer+ cells with induced resistance to MTIC was similar to that in Mer- cells. Neither adenovirus 5 nor HSV-1 infection induced ATase activity in Mer- cells. This indicates that resistance to the toxic effects of methylating agents is not invariably associated with high levels of ATase activity in human melanoma cells. Furthermore, while induction of the Mer+ phenotype from Mer- cells was usually accompanied by the recovery of ATase activity, induced Mer+ cells had less proficient repair than natural Mer+ cells, as judged quantitatively by slightly lower cellular resistance and qualitatively by deficient HCR response for HSV-1. These results suggest that the Mer- and induced Mer+ cells lack an ATase-independent DNA repair mechanism. No differences in MTIC-induced DNA repair synthesis or strand breaks were found between the Mer-, natural Mer+, and induced Mer+ phenotypes. However, UV-induced DNA repair synthesis was higher in the natural Mer+ than in the Mer- or induced Mer+ cells, both of which had increased cellular sensitivity to the antimetabolites methotrexate and hydroxyurea

  19. All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

    KAUST Repository

    So, Hongyun; Lee, Kunwoo; Murthy, Niren; Pisano, Albert P

    2014-01-01

    This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

  20. All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

    KAUST Repository

    So, Hongyun

    2014-11-24

    This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

  1. On DNA synthesis during C14O2 assimilation by peas seedlings

    International Nuclear Information System (INIS)

    Karimov, Kh.Kh.; Nikolaeva, M.I.

    1976-01-01

    In this article authors try to determine how much p articipate t hephotosynthesis in the new formation of DNA seedlings, depends this processfrom the light and realize at this the synthesis DNA in chloroplasts

  2. Test of models for replication of SV40 DNA following UV irradiation

    International Nuclear Information System (INIS)

    Barnett, S.W.

    1983-01-01

    The replication of SV40 DNA immediately after irradiation of infected monkey cells has been examined. SV40 DNA synthesis is inhibited in a UV fluence-dependent fashion, and the synthesis of completely replicated (Form I) SV40 molecules is more severely inhibited than is total SV40 DNA synthesis. Two models for DNA replication-inhibition have been tested. Experimental results have been compared to those predicted by mathematical models derived to describe two possible molecular mechanisms of replication inhibition. No effect of UV irradiation on the uptake and phosphorylation of 3 H-thymidine nor on the size of the intracellular deoxythymidine triphosphate pool of SV40-infected cells have been observed, validating the use of 3 H-thymidine incorporation as a measure of DNA synthesis in this system. In vitro studies have been performed to further investigate the mechanism of dimer-specific inhibition of completion of SV40 DNA synthesis observed in in vivo. The results of these studies are consistent with a mechanism of discontinuous synthesis past dimer sites, but it is equally possible that the mechanism of DNA replication of UV-damaged DNA in the in vitro system is different from that which occurs in vivo

  3. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis

    DEFF Research Database (Denmark)

    Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy

    2017-01-01

    stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks...

  4. Cell proliferation and DNA dependent DNA polymerase estimation in acute lymphoblastic leukaemia during treatment with prednisone and vincristine

    International Nuclear Information System (INIS)

    Lange Wantzin, G.

    1979-01-01

    The presence of DNA polymerase and primer-template DNA in lymphoblast nuclei by measuring the in vitro incorporation of 3 H-thymidine-5'-triphosphate ( 3 H-TTP) was studied in 10 patients with acute lymphoblastic leukemia. Protein synthesis and various other cytokinetic parameters were also studied. After prednisone (P) administration a marked decrease in 3 H-TTP labelling index ( 3 H-TTP LI) was apparent together with an inhibition of 3 H-leucine incorporation ( 3 H-LEU LI) into lymphoblasts. A moderate decrease in 3 H-TDR labelling index ( 3 H-TDR LI) and a later decrease in mitotic index (MI) were seen. Single cell DNA measurements showed a depletion of 3 H-TDR labelled lymphoblasts in early part of S-phase apparent at 24 h lasting up to 54 h after P administration. Vincristine given as a flash injection later in the study period caused an immediate rise of the MI, at the same time the P induced decline in 3 H-TTP LI, 3 H-TDR LI and 3 H-LEU LI were continued in most patients. P is thought to damage the cells both in and outside the cell cycle. In the cell cycle the effect of P is an arresting effect in G 1 . (author)

  5. Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

    International Nuclear Information System (INIS)

    Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

    2005-01-01

    Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

  6. Assay for Epstein--Barr virus based on stimulation of DNA systhesis in mixed leukocytes from human umbilical cord blood

    International Nuclear Information System (INIS)

    Robinson, J.; Miller, G.

    1975-01-01

    Relationships between the rate of DNA synthesis in cultured human umbilical cord leukocytes and the multiplicity of added Epstein-Barr virus (EBV) were studied. At low multiplicities of approximately 0.1 transforming units/cell (approximately 10 physical particles/cell), inoculated cultures demonstrated increased rates of DNA synthesis, by comparison to uninoculated cultures, 3 days after inoculation. Stimulation of DNA synthesis was evident at progressively longer intervals after inoculations of 10-fold dilutions of virus. The rate of DNA synthesis, determined by short [ 3 H]thymidine pulses, reflected as small as twofold changes in multiplicity and thus can serve as a quantitative assay for the virus. Changes in the rate of DNA synthesis were evident before increases in cell number or alteration in morphology. Stimulation of DNA synthesis in umbilical cord leukocytes was inhibited by treatment of EBV with antibody and also in graded fashion, by progressive doses of uv irradiation to the virus. Induction of DNA synthesis by EBV was serum dependent. Estimates of the number of cells transformed were obtained by extrapolation from a standard curve relating known numbers of transformed cells to [ 3 H]thymidine incorporation and also by cloning cells after exposure to virus. At the low multiplicities of infection used in these experiments approximately 0.04 to 0.002 of the total cellular population was transformed. The high efficiency of cell transformation by EBV by comparison to other DNA tumor viruses is emphasized

  7. Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

    International Nuclear Information System (INIS)

    Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

    1989-01-01

    Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector λpSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB + clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA + transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB + and lysA + . The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes

  8. Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells

    International Nuclear Information System (INIS)

    Freed, J.J.; Hoess, R.H.; Angelosanto, F.A.; Massey, H.C. Jr.

    1979-01-01

    Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (D 0 ) in the range 1.5-1.7 Jm -2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dsub(q)), the value for exponentially multiplying diploid cells (3.0 Jm -2 ) being higher than that found for haploids (1.2 Jm -2 ). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromoatographically during up to 48 h incubation in the dark and since autoradiographically detected 'unscheduled DNA synthesis' was limited to a 2-fold increase saturated at 10 Jm -2 . These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield. (Auth.)

  9. DNA hydrogel as a template for synthesis of ultrasmall gold nanoparticles for catalytic applications.

    Science.gov (United States)

    Zinchenko, Anatoly; Miwa, Yasuyuki; Lopatina, Larisa I; Sergeyev, Vladimir G; Murata, Shizuaki

    2014-03-12

    DNA cross-linked hydrogel was used as a matrix for synthesis of gold nanoparticles. DNA possesses a strong affinity to transition metals such as gold, which allows for the concentration of Au precursor inside a hydrogel. Further reduction of HAuCl4 inside DNA hydrogel yields well dispersed, non-aggregated spherical Au nanoparticles of 2-3 nm size. The average size of these Au nanoparticles synthesized in DNA hydrogel is the smallest reported so far for in-gel metal nanoparticles synthesis. DNA hybrid hydrogel containing gold nanoparticles showed high catalytic activity in the hydrogenation reaction of nitrophenol to aminophenol. The proposed soft hybrid material is promising as environmentally friendly and sustainable material for catalytic applications.

  10. Synthesis of a multi-functional DNA nanosphere barcode system for direct cell detection.

    Science.gov (United States)

    Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2017-09-28

    Nucleic acid-based technologies have been applied to numerous biomedical applications. As a novel material for target detection, DNA has been used to construct a barcode system with a range of structures. This paper reports multi-functionalized DNA nanospheres (DNANSs) by rolling circle amplification (RCA) with several functionalized nucleotides. DNANSs with a barcode system were designed to exhibit fluorescence for coding enhanced signals and contain biotin for more functionalities, including targeting through the biotin-streptavidin (biotin-STA) interaction. Functionalized deoxynucleotide triphosphates (dNTPs) were mixed in the RCA process and functional moieties can be expressed on the DNANSs. The anti-epidermal growth factor receptor antibodies (anti-EGFR Abs) can be conjugated on DNANSs for targeting cancer cells specifically. As a proof of concept, the potential of the multi-functional DNANS barcode was demonstrated by direct cell detection as a simple detection method. The DNANS barcode provides a new route for the simple and rapid selective recognition of cancer cells.

  11. Câncer e agentes antineoplásicos ciclo-celular específicos e ciclo-celular não específicos que interagem com o DNA: uma introdução Cancer and cell cicle-specific and cell cicle nonspecific anticancer DNA-interactive agents: an introduction

    Directory of Open Access Journals (Sweden)

    Vera Lúcia de Almeida

    2005-02-01

    Full Text Available The chemotherapy agents against cancer may be classified as "cell cycle-specific" or "cell cycle-nonspecific". Nevertheless, several of them have their biological activity related to any kind of action on DNA such as: antimetabolic agents (DNA synthesis inhibition, inherently reactive agents (DNA alkylating electrophilic traps for macromolecular nucleophiles from DNA through inter-strand cross-linking - ISC - alkylation and intercalating agents (drug-DNA interactions inherent to the binding made due to the agent penetration in to the minor groove of the double helix. The earliest and perhaps most extensively studied and most heavily employed clinical anticancer agents in use today are the DNA inter-strand cross-linking agents.

  12. A unique DNA found in post-mitochondrial fraction from Ehrlich ascites tumor cells

    International Nuclear Information System (INIS)

    Guimaraes, R.C.; Bloch, D.P.

    1982-01-01

    A DNA found in post-mitochondrial fractions from Ehrlich ascites cells, comprising 0.2% of the total cellular DNA, is partially characterized. It appears in cytoplasmic homogenates as a 14.6 S molecule, and is eluted from hydroxyapatite with 0.24 M sodium phosphate buffer. Its Cs 2 SO 4 buoyant density is lower than Erlich ascites tumor nuclear DNA and it has low dG+dC content, as determined by chromatography of hydrolysates of 32 P-labelled DNA. It is enriched in sequences which reassociate rapidly in the presence of excess nuclear DNA. It can be used as promoter for DNA synthesis by an endogenous DNA-dependent DNA polymerase found in association with the post-mitochondrial preparations. It is found to be associated with newly incorporated radioactivity following incubation in vitro with labelled UTP. Its localization in situ has not yet been attempled. It is thought to represent viral A-type particle associated, or plasma membrane associated DNA. (author) [pt

  13. Size and frequency of gaps in newly synthesized DNA of xeroderma pigmentosum human cells irradiated with ultraviolet light

    International Nuclear Information System (INIS)

    Meneghini, R.; Cordeiro-Stone, M.; Schumacher, R.I.

    1981-01-01

    Native newly synthesized DNA from human cells (xeroderma pigmentosum type) irradiated with ultraviolet light releases short pieces of DNA (L-DNA) when incubated with the single-strand specific S 1 nuclease. This is not observed in the case of unirradiated cells. Previous experiments had shown that the L-DNA resulted from the action of S 1 nuclease upon gaps, i.e., single-stranded DNA discontinuities in larger pieces of double-stranded DNA. We verified that the duplex L-DNA, that arises from the inter-gap regions upon S 1 nuclease treatment, has a size which approximates the distance between two pyrimidine dimers on the same strand. A method was devised to measure the size of the gaps. These parameters have been considered in the proposition of a model for DNA synthesis on a template containing pyrimidine dimers

  14. Cytometry of DNA Replication and RNA Synthesis: Historical Perspective and Recent Advances Based on “Click Chemistry”

    OpenAIRE

    Darzynkiewicz, Zbigniew; Traganos, Frank; Zhao, Hong; Halicka, H. Dorota; Li, Jiangwei

    2011-01-01

    This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of 3H- or 14C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G1, S, G2, and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of 3H-uridine incorporation and RNA c...

  15. Stwl modifies chromatin compaction and is required to maintain DNA integrity in the presence of perturbed DNA replication

    NARCIS (Netherlands)

    Yi, X.; Vries, de H.I.; Siudeja, K.; Rana, A.; Lemstra, W.; Brunsting, J.F.; Kok, R.J.M.; Smulders, Y.M.; Schaefer, M.; Dijk, F.; Shang, Y.F.; Eggen, B.J.L.; Kampinga, H.H.; Sibon, O.C.M.

    2009-01-01

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent

  16. Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

    NARCIS (Netherlands)

    Yi, Xia; Vries, Hilda I. de; Siudeja, Katarzyna; Rana, Anil; Lemstra, Willy; Brunsting, Jeanette F.; Kok, Rob M.; Smulders, Yvo M.; Schaefer, Matthias; Dijk, Freark; Shang, Yongfeng; Eggen, Bart J.L.; Kampinga, Harm H.; Sibon, Ody C.M.

    Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent

  17. DNA repair in mutagen-injured higher plants

    International Nuclear Information System (INIS)

    Veleminsky, J.; Gichner, T.

    1978-01-01

    Data are summarized proving the occurrence of photoreactivation of UV-induced pyrimidine dimers in cells of Nicotiana tabucum, Gingko and carrot, the excision of dimers in cells of Nicotiana tabacum, Gingko and carrot, the excision of dimers in protoplasts of carrot and in embryos of Lathyrus sativus, and the repair of DNA single-strand breaks induced in carrot protoplasts and barley embryonic cells by ionizing radiation. In irradiated barley embryos the unscheduled DNA synthesis and higher accessibility of induced primers to DNA polymerase I of E. coli were observed preferentially in G 1 cells with diffused chromatin. These reactions were inhibited by caffeine and EDTA. Unscheduled DNA synthesis was also observed in synchronized irradiated root cuttings of Vicia faba and in barley embryos treated with 4-nitroquinoline oxide, the latter being inhibited by caffeine and hydroxyurea. Repair synthesis was also established in barley embryos treated with mutagenic N-methyl-N-nitrosourea under conditions that postponed the onset of germination after the treatment. The same conditions enhanced the repair of DNA single-strand breaks induced by this mutagen and several other monofunctional alkylating compounds. From tissues of barley and of Phaseolus multiflorus, endonucleases for apurinic sites were isolated and characterized. Some of them are located in chromatin, others in chloroplasts. The relation between DNA repair and genetic effects of mutagens in higher plants is also discussed. (Auth.)

  18. A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

    Science.gov (United States)

    Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

    2017-11-01

    Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  19. Carcinogen-induced damage to DNA

    International Nuclear Information System (INIS)

    Strauss, B.; Altamirano, M.; Bose, K.; Sklar, R.; Tatsumi, K.

    1979-01-01

    Human cells respond to carcinogen-induced damage in their DNA in at least two ways. The first response, excision repair, proceeds by at least three variations, depending on the nature of the damage. Nucleotide excision results in relatively large repair patches but few free DNA breaks, since the endonuclease step is limiting. Apurinic repair is characterized by the appearance of numerous breaks in the DNA and by short repair patches. The pathways behave as though they function independently. Lymphoic cells derived from a xeroderma pigmentosum complementation group C patient are deficient in their ability to perform nucleotide excision and also to excise 6 methoxyguanine adducts, but they are apurinic repair competent. Organisms may bypass damage in their DNA. Lymphoblastoid cells, including those derived from xeroderma pigmentosum treated with 3 H-anti-BPDE, can replicate their DNA at low doses of carcinogen. Unexcised 3 H is found in the light or parental strand of the resulting hybrid DNA when replication occurs in medium with BrdUrd. This observation indicates a bypass reaction occurring by a mechanism involving branch migration at DNA growing points. Branch migration in DNA preparations have been observed, but the evidence is that most occurs in BrdUrd-containing DNA during cell lysis. The measurement of the bifilarly substituted DNA resulting from branch migration is a convenient method of estimating the proportion of new synthesis remaining in the vicinity of the DNA growing point. Treatment with carcinogens or caffeine results in accumulation of DNA growing points accompanied by the synthesis of shortened pieces of daughter DNA

  20. Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes

    International Nuclear Information System (INIS)

    Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G.; Okuno, Yasuyoshi

    2009-01-01

    High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 μM MTF and 50 μM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 μM MTF and 100-500 μM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver

  1. Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

    Science.gov (United States)

    Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G; Okuno, Yasuyoshi

    2009-04-05

    High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

  2. CdS nanowires formed by chemical synthesis using conjugated single-stranded DNA molecules

    Science.gov (United States)

    Sarangi, S. N.; Sahu, S. N.; Nozaki, S.

    2018-03-01

    CdS nanowires were successfully grown by chemical synthesis using two conjugated single-stranded (ss) DNA molecules, poly G (30) and poly C (30), as templates. During the early stage of the synthesis with the DNA molecules, the Cd 2+ interacts with Poly G and Poly C and produces the (Cd 2+)-Poly GC complex. As the growth proceeds, it results in nanowires. The structural analysis by grazing angle x-ray diffraction and transmission electron microscopy confirmed the zinc-blende CdS nanowires with the growth direction of . Although the nanowires are well surface-passivated with the DNA molecules, the photoluminescence quenching was caused by the electron transfer from the nanowires to the DNA molecules. The quenching can be used to detect and label the DNAs.

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

    International Nuclear Information System (INIS)

    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

  4. Radiation induced formation of giant cells (Saccharomyces uvarum). Pt. 1

    Energy Technology Data Exchange (ETDEWEB)

    Baumstark-Khan, C; Schnitzler, L; Rink, H

    1984-02-01

    X-irradiated yeast cells (Saccharomyces uvarum) grown in liquid media stop mitosis and form giant cells. Chitin ring formation, being a prerequisite for cell separation, was studied by fluorescence microscopy using Calcofluor White, a chitin specific dye. Experiments with inhibitors of DNA synthesis (hydroxyurea) and chitin synthesis (polyoxin D) demonstrate chitin ring formation to be dependent on DNA synthesis, whereas bud formation is independent of DNA synthesis and chitin ring formation respectively. Basing on these results the formation of X-ray induced giant cells implies one DNA replication which in turn induces the formation of only one chitin ring between mother cell and giant bud. Obviously no septum can be formed. Thus cell separation does not occur, but the bud already formed, produces another bud demonstrating that bud formation itself is independent of DNA synthesis.

  5. Control of density-dependent, cell state-specific signal transduction by the cell adhesion molecule CEACAM1, and its influence on cell cycle regulation

    International Nuclear Information System (INIS)

    Scheffrahn, Inka; Singer, Bernhard B.; Sigmundsson, Kristmundur; Lucka, Lothar; Oebrink, Bjoern

    2005-01-01

    Growth factor receptors, extracellular matrix receptors, and cell-cell adhesion molecules co-operate in regulating the activities of intracellular signaling pathways. Here, we demonstrate that the cell adhesion molecule CEACAM1 co-regulates growth-factor-induced DNA synthesis in NBT-II epithelial cells in a cell-density-dependent manner. CEACAM1 exerted its effects by regulating the activity of the Erk 1/2 MAP kinase pathway and the expression levels of the cyclin-dependent kinase inhibitor p27 Kip1 . Interestingly, both inhibitory and stimulatory effects were observed. Confluent cells continuously exposed to fetal calf serum showed little Erk activity and DNA synthesis compared with sparse cells. Under these conditions, anti-CEACAM1 antibodies strongly stimulated Erk activation, decreased p27 expression, and induced DNA synthesis. In serum-starved confluent cells, re-addition of 10% fetal calf serum activated the Erk pathway, decreased p27 expression, and stimulated DNA synthesis to the same levels as in sparse cells. Under these conditions anti-CEACAM1 antibodies de-activated Erk, restored the level of p27, and inhibited DNA synthesis. These data indicate that CEACAM1 mediates contact inhibition of proliferation in cells that are constantly exposed to growth factors, but co-activates growth-factor-induced proliferation in cells that have been starved for growth factors; exposure to extracellular CEACAM1 ligands reverts these responses

  6. Repair of abasic sites in DNA

    Energy Technology Data Exchange (ETDEWEB)

    Dianov, Grigory L.; Sleeth, Kate M.; Dianova, Irina I.; Allinson, Sarah L

    2003-10-29

    Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase {beta} adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase {delta}/{epsilon} and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase {delta}/{epsilon} is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.

  7. Low-intensity pulsed ultrasound stimulates cell proliferation, proteoglycan synthesis and expression of growth factor-related genes in human nucleus pulposus cell line

    Directory of Open Access Journals (Sweden)

    Y Kobayashi

    2009-06-01

    Full Text Available Low-intensity pulsed ultrasound (LIPUS stimulation has been shown to effect differentiation and activation of human chondrocytes. A study involving stimulation of rabbit disc cells with LIPUS revealed upregulation of cell proliferation and proteoglycan (PG synthesis. However, the effect of LIPUS on human nucleus pulposus cells has not been investigated. In the present study, therefore, we investigated whether LIPUS stimulation of a human nucleus pulposus cell line (HNPSV-1 exerted a positive effect on cellular activity. HNPSV-1 cells were encapsulated in 1.2% sodium alginate solution at 1x105 cells/ml and cultured at 10 beads/well in 6-well plates. The cells were stimulated for 20 min each day using a LIPUS generator, and the effects of LIPUS were evaluated by measuring DNA and PG synthesis. Furthermore, mRNA expression was analyzed by cDNA microarray using total RNA extracted from the cultured cells. Our study revealed no significant difference in cell proliferation between the control and the ultrasound treated groups. However, PG production was significantly upregulated in HNPSV cells stimulated at intensities of 15, 30, 60, and 120 mW/cm2 compared with the control. The results of cDNA array showed that LIPUS significantly stimulated the gene expression of growth factors and their receptors (BMP2, FGF7, TGFbetaR1 EGFRF1, VEGF. These findings suggest that LIPUS stimulation upregulates PG production in human nucleus pulposus cells by the enhancement of several matrix-related genes including growth factor-related genes. Safe and non-invasive stimulation using LIPUS may be a useful treatment for delaying the progression of disc degeneration.

  8. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    International Nuclear Information System (INIS)

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-01-01

    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: [ 3 H]thymidine incorporation decreased by 80% and [ 14 C]leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of [ 3 H]histidine/[ 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

  10. Human cytomegalovirus uracil DNA glycosylase associates with ppUL44 and accelerates the accumulation of viral DNA

    Directory of Open Access Journals (Sweden)

    Dixon Melissa

    2005-07-01

    Full Text Available Abstract Background Human cytomegalovirus UL114 encodes a uracil-DNA glycosylase homolog that is highly conserved in all characterized herpesviruses that infect mammals. Previous studies demonstrated that the deletion of this nonessential gene delays significantly the onset of viral DNA synthesis and results in a prolonged replication cycle. The gene product, pUL114, also appears to be important in late phase DNA synthesis presumably by introducing single stranded breaks. Results A series of experiments was performed to formally assign the observed phenotype to pUL114 and to characterize the function of the protein in viral replication. A cell line expressing pUL114 complemented the observed phenotype of a UL114 deletion virus in trans, confirming that the observed defects were the result of a deficiency in this gene product. Stocks of recombinant viruses without elevated levels of uracil were produced in the complementing cells; however they retained the phenotype of poor growth in normal fibroblasts suggesting that poor replication was unrelated to uracil content of input genomes. Recombinant viruses expressing epitope tagged versions of this gene demonstrated that pUL114 was expressed at early times and that it localized to viral replication compartments. This protein also coprecipitated with the DNA polymerase processivity factor, ppUL44 suggesting that these proteins associate in infected cells. This apparent interaction did not appear to require other viral proteins since ppUL44 could recruit pUL114 to the nucleus in uninfected cells. An analysis of DNA replication kinetics revealed that the initial rate of DNA synthesis and the accumulation of progeny viral genomes were significantly reduced compared to the parent virus. Conclusion These data suggest that pUL114 associates with ppUL44 and that it functions as part of the viral DNA replication complex to increase the efficiency of both early and late phase viral DNA synthesis.

  11. Cell proliferation and DNA dependent DNA polymerase estimation in acute lymphoblastic leukaemia during treatment with prednisone and vincristine

    Energy Technology Data Exchange (ETDEWEB)

    Lange Wantzin, G [Rigshospitalet, Copenhagen (Denmark)

    1979-01-01

    The presence of DNA polymerase and primer-template DNA in lymphoblast nuclei by measuring the in vitro incorporation of /sup 3/H-thymidine-5'-triphosphate (/sup 3/H-TTP) was studied in 10 patients with acute lymphoblastic leukemia. Protein synthesis and various other cytokinetic parameters were also studied. After prednisone (P) administration a marked decrease in /sup 3/H-TTP labelling index (/sup 3/H-TTP LI) was apparent together with an inhibition of /sup 3/H-leucine incorporation (/sup 3/H-LEU LI) into lymphoblasts. A moderate decrease in /sup 3/H-TDR labelling index (/sup 3/H-TDR LI) and a later decrease in mitotic index (MI) were seen. Single cell DNA measurements showed a depletion of /sup 3/H-TDR labelled lymphoblasts in early part of S-phase apparent at 24 h lasting up to 54 h after P administration. Vincristine given as a flash injection later in the study period caused an immediate rise of the MI, at the same time the P induced decline in /sup 3/H-TTP LI, /sup 3/H-TDR LI and /sup 3/H-LEU LI were continued in most patients. P is thought to damage the cells both in and outside the cell cycle. In the cell cycle the effect of P is an arresting effect in G/sub 1/.

  12. Histoautoradiographic and liquid scintillometric studies on DNA synthesis in the liver, kidneys, spleen and tongue after bilateral adrenalectomy in rats

    International Nuclear Information System (INIS)

    Schneider, A.

    1981-01-01

    Historadiographies and liquid scintillometries were carried out in 163 male Wistar rats in order to determine the effects of bilateral adrenalectomy on DNA synthesis in the liver, kidneys, spleen, and tongue. Both DNA synthesis and mitotic index are significantly increased from the 1st day p.o. onwards, with broad synthesis peaks between the 2nd and the 4th day. The intensity of DNA synthesis shows a gradual decrease with increasing duration of the experiment. In contrast to the adrenalectonized animals, the synthesis rate and mitotic index in the organs of sham-operated animals were significantly lower, although enhanced proliferation was observed after surgery. The enhanced DNA synthesis after bilateral adrenalectomy is interpreted in terms of a disinhibition; corticosteroids are assumed to play a key role. The effects of bilateral adrenalectromy on untreated organs are not organ-specific. The highest synthesis rate was observed in the tubular epithelia of the convoluted main parts, while the DNA synthesis in the tongue. The findings of autoradiography and liquid scintillometry are well correlated. (orig./MG) [de

  13. Design and synthesis of DNA four-helix bundles

    Energy Technology Data Exchange (ETDEWEB)

    Rangnekar, Abhijit; Gothelf, Kurt V [Department of Chemistry, Centre for DNA Nanotechnology (CDNA) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C (Denmark); LaBean, Thomas H, E-mail: kvg@chem.au.dk, E-mail: thl@cs.duke.edu [Department of Chemistry, Duke University, Durham, NC 27708 (United States)

    2011-06-10

    The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

  14. Design and synthesis of DNA four-helix bundles

    International Nuclear Information System (INIS)

    Rangnekar, Abhijit; Gothelf, Kurt V; LaBean, Thomas H

    2011-01-01

    The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

  15. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity ...

    Indian Academy of Sciences (India)

    s12039-016-1125-x. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity of lanthanide(III) complexes of 2-benzoylpyridine acetylhydrazone. KARREDDULA RAJA, AKKILI SUSEELAMMA and KATREDDI HUSSAIN REDDY. ∗.

  16. Radiation induced formation of giant cells (Saccharomyces uvarum). Pt. 1

    International Nuclear Information System (INIS)

    Baumstark-Khan, C.; Schnitzler, L.; Rink, H.

    1984-01-01

    X-irradiated yeast cells (Saccharomyces uvarum) grown in liquid media stop mitosis and form giant cells. Chitin ring formation, being a prerequisite for cell separation, was studied by fluorescence microscopy using Calcofluor White, a chitin specific dye. Experiments with inhibitors of DNA synthesis (hydroxyurea) and chitin synthesis (polyoxin D) demonstrate chitin ring formation to be dependent on DNA synthesis, whereas bud formation is independent of DNA synthesis and chitin ring formation respectively. Basing on these results the formation of X-ray induced giant cells implies one DNA replication which in turn induces the formation of only one chitin ring between mother cell and giant bud. Obviously no septum can be formed. Thus cell separation does not occur, but the bud already formed, produces another bud demonstrating that bud formation itself is independent of DNA synthesis. (orig.)

  17. Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

    Science.gov (United States)

    Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

    2017-03-15

    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.

  18. Studies on the relationship between the cancer chemotherapeutic agent, hydroxyurea, and DNA repair in mammalian cells

    International Nuclear Information System (INIS)

    Katz, E.J.

    1988-01-01

    To examine the possibility that manipulating DNA repair might lessen drug resistance, we investigated whether depletion of the thymidine triphosphate (TTP) pool or administration of hydroxyurea could interfere with the ability of confluent normal human skin fibroblasts to repair ultraviolet irradiation-induced DNA damage. A method was developed for the quantitation of cellular TTP pools by labeling them with [ 3 H]thymidine. The addition of hydroxyurea, either simultaneously with [ 3 H]thymidine or two hours later, resulted in a dose- and time-dependent increase in the [ 3 H]TTP pool. The capacity of these cells to carry out DNA repair was quantitated by their ability to perform repair replication synthesis of DNA after exposure to ultraviolet irradiation. This radiation produces thymine dimers in DNA, which are repaired by the nucleotide excision repair pathway. The experimental protocol resulted in an 8-10-fold reduction in the [ 3 H]TTP pool. Saturating levels of DNA repair synthesis were observed under these conditions, with no further diminution of the already reduced [ 3 H]TTP pool. Repair replication and [ 3 H]TTP pool measurements were identical in cultures treated with 10 mM hydroxyurea and in those not exposed to the drug

  19. Effects of an extract from the sea squirt Ecteinascidia turbinata on DNA synthesis and excision repair in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dunn, W.C.; Carrier, W.L.; Regan, J.D.

    1982-01-01

    An aqueous ethanol extract from the marine tunicate species Ecteinascidia turbinata was studied to determine its effect on semiconservative DNA synthesis in human skin fibroblast cultures as measured by (/sup 3/H) thymidine uptake in acid-insoluble cell fractions. In addition, the effect of this extract on DNA excision repair in ultraviolet light (254 nm) irradiated fibroblasts was measured by the bromodeoxyuridine photolysis assay, thymine dimer chromatography, and DNA single-strand break analysis on alkaline sucrose gradients. Repair inhibition was accompanied by an accumulation of single-strand DNA breaks which was enhanced by the addtion of 2 mM hydroxyurea. These results are discussed with respect to a mechanism of action of the marine tunicate extract at the level of DNA polymerases and are contrasted with previously studied inhibitory mechanisms of arabinofuranosyl nucleosides.

  20. Estimation of pre-meiotic DNA synthesis period in the dog spermatozoa

    International Nuclear Information System (INIS)

    Ghosal, S.K.; Bandyopadhyay, T.; De, S.; Beauregard, L.J.

    1976-01-01

    About 10 μCi of 3 H-thymidine was injected into each of 4 arbitarary sites in each testis of 6 dogs. Biopsies were taken at 4-hour intervals coverning a period from 20.0 to 22.2 days post-injection. Kinetics of labelled spermatocytes was followed employing Kodak NTB-3 emulsion to conventionally prepared air-dried slides. The technique for calculating pre-meiosis DNA synthesis duration is same as that for estimating S period in mitotic cells. Current investigation suggests that the mean duration of pre-meiotic S period of Canine spermatocytes is 20.4 hrs as compared to 29 and 40 hrs in spermatocytes of mouse and golden hamster respectively. (author)

  1. Radiation effect and response of DNA synthesis in lymphocytes induced by low dose irradiation

    International Nuclear Information System (INIS)

    Zhao Yujie; Su Liaoyuan; Zou Huawei; Kong Xiangrong

    1999-01-01

    The ability of DNA synthesis in lymphocytes were measured by using 3 H-TdR incorporation method. This method was used to observe the damage of lymphocytes irradiated by several challenge doses (0.5-0.8 Gy) and adaptive response induced by previous low dose irradiation. The results show that DNA synthesis was inhibited by challenge dose of radiation and was adapted by previous 0.048 Gy irradiation

  2. Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins.

    Science.gov (United States)

    Sparwasser, T; Vabulas, R M; Villmow, B; Lipford, G B; Wagner, H

    2000-12-01

    Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co-stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen-reactive CD4 T helper cells via CD40 signaling. Once activated, antigen-presenting DC may activate CD8 T cell responses in a T helper cell-independent fashion. Using immunostimulatory CpG-oligonucleotides (ODN) mimicking bacterial CpG-DNA, we tested whether CpG-DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen-presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen-specific CD8 cytotoxic T lymphocyte (CTL)-mediated responses becomes initiated in wild-type as well as T helper cell-deficient mice, provided soluble protein and CpG-ODN are draining into the same lymph node. Specifically we show that CpG-ODN trigger antigen-presenting immature DC within the draining lymph node to acutely up-regulate co-stimulatory molecules and produce IL-12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.

  3. DNA-templated antibody conjugation for targeted drug delivery to cancer cells

    DEFF Research Database (Denmark)

    Liu, Tianqiang

    2016-01-01

    -templated organic synthesis due to the wide existence of the 3-histidine cluster in most wild-type proteins. In this thesis, three projects that relate to targeted drug delivery to cancer cells based on the DTPC method is described. The first project was a delivery system which uses transferrin as the targeting....... The study shows that DNA is a highly useful tool for the assembly of proteins with potential therapeutic applications. The DNA-templated protein conjugation shows a promising application in constructing antibody-toxin conjugates or antibody-drug conjugates. In addition, DNA strands used for antibody...... either antibody engineering or special expression systems and are both time and labor consuming. To avoid the drawbacks caused by conventional chemical modification and recombinant methodologies, an ideal site specific conjugation technique would use natural amino acid residues to the protein by a new...

  4. Programmable autonomous synthesis of single-stranded DNA

    Science.gov (United States)

    Kishi, Jocelyn Y.; Schaus, Thomas E.; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

    2018-02-01

    DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

  5. Mechanisms of DNA uptake by cells

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Three categories of cellular uptake of DNA can be distinguished. First, in the highly transformable bacteria, such as Diplococcus pneumoniae, Haemophilus influenzae and Bacillus subtilis, elaborate mechanisms of DNA transport have evolved, presumably for the purpose of genetic exchange. These mechanisms can introduce substantial amounts of DNA into the cell. Second, methods have been devised for the forced introduction of DNA by manipulation of bacterial cells under nonphysiological conditions. By such means small but significant amounts of DNA have been introduced into various bacteria, including Escherichia coli. Third, mammalian cells are able to take up biologically active DNA. This has been most clearly demonstrated with viral DNA, although the mechanism of uptake is not well understood. The intention, here, is to survey current understanding of the various mechanisms of DNA uptake. A review of experience with the bacterial systems may throw some light on the mammalian system and lead to suggestions for enhancing DNA uptake by mammalian cells.

  6. Permeability changes and incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of Chinese hamsters affected by MEA and low temperature

    International Nuclear Information System (INIS)

    Ermekova, V.M.; Kondakova, N.V.; Levitman, M.Kh.; Saugabaeva, K.M.; Ehjdus, L.Kh.

    1976-01-01

    Action of MEA and low temperature (20degC) on the incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of chinese hamsters has been studied. It has been found that each of the above-mentioned factors equally decreases the label uptake into the cell and DNA. It is concluded that MEA and low temperature do not substantially influence the rate of DNA synthesis

  7. 1-{beta}-D-arabinofuranosylcytosine is cytotoxic in quiescent normal lymphocytes undergoing DNA excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Yamauchi, Takahiro; Kawai, Yasukazu; Ueda, Takanori [Fukui Medical Univ., Matsuoka (Japan)

    2002-12-01

    We have sought to clarify the potential activity of the S-phase-specific antileukemic agent 1-{beta}-D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non-sensitive to nucleoside analogues. It was hypothesized that the combination of ara-C with DNA damaging agents that initiate DNA repair will expand ara-C cytotoxicity to non-cycling cells. The repair kinetics, which included incision of damaged DNA, gap-filling by DNA synthesis and rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara-C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara-C-mediated inhibition of the repair processes was concentration-dependent, with the effect peaking at 10{mu}M. The combination of ara-C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara-C. In conclusion, ara-C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara-C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy. (author)

  8. 1-β-D-arabinofuranosylcytosine is cytotoxic in quiescent normal lymphocytes undergoing DNA excision repair

    International Nuclear Information System (INIS)

    Yamauchi, Takahiro; Kawai, Yasukazu; Ueda, Takanori

    2002-01-01

    We have sought to clarify the potential activity of the S-phase-specific antileukemic agent 1-β-D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non-sensitive to nucleoside analogues. It was hypothesized that the combination of ara-C with DNA damaging agents that initiate DNA repair will expand ara-C cytotoxicity to non-cycling cells. The repair kinetics, which included incision of damaged DNA, gap-filling by DNA synthesis and rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara-C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara-C-mediated inhibition of the repair processes was concentration-dependent, with the effect peaking at 10μM. The combination of ara-C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara-C. In conclusion, ara-C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara-C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy. (author)

  9. Correlation between DNA repair of embryonic fibroblasts and different life span of 3 inbred mouse strains

    Energy Technology Data Exchange (ETDEWEB)

    Paffenholz, V.

    1978-02-01

    Primary mouse fibroblast cultures were established from 10 day old embryos of 3 inbred strains with a genetically determined different life expectancy. The capacity for unscheduled DNA synthesis following uv irradiation was studied in these cells at various passage levels of the in vitro ageing process. The mouse fibroblasts show considerable repair synthesis corresponding to the duration of exposure time. The capacity for induction of unscheduled DNA synthesis was different in the cells of each strain and correlated to the natural life span of the animal. In each case, however, the ability to perform repair synthesis was subjected to an age-associated decline, although semiconservative DNA synthesis and proliferative potential of the cell was not changed until the cultures entered phase III passages.

  10. Self-assembled catalytic DNA nanostructures for synthesis of para-directed polyaniline.

    Science.gov (United States)

    Wang, Zhen-Gang; Zhan, Pengfei; Ding, Baoquan

    2013-02-26

    Templated synthesis has been considered as an efficient approach to produce polyaniline (PANI) nanostructures. The features of DNA molecules enable a DNA template to be an intriguing template for fabrication of emeraldine PANI. In this work, we assembled HRP-mimicking DNAzyme with different artificial DNA nanostructures, aiming to manipulate the molecular structures and morphologies of PANI nanostructures through the controlled DNA self-assembly. UV-vis absorption spectra were used to investigate the molecular structures of PANI and monitor kinetic growth of PANI. It was found that PANI was well-doped at neutral pH and the redox behaviors of the resultant PANI were dependent on the charge density of the template, which was controlled by the template configurations. CD spectra indicated that the PANI threaded tightly around the helical DNA backbone, resulting in the right handedness of PANI. These reveal the formation of the emeraldine form of PANI that was doped by the DNA. The morphologies of the resultant PANI were studied by AFM and SEM. It was concluded from the imaging and spectroscopic kinetic results that PANI grew preferably from the DNAzyme sites and then expanded over the template to form 1D PANI nanostructures. The strategy of the DNAzyme-DNA template assembly brings several advantages in the synthesis of para-coupling PANI, including the region-selective growth of PANI, facilitating the formation of a para-coupling structure and facile regulation. We believe this study contributes significantly to the fabrication of doped PANI nanopatterns with controlled complexity, and the development of DNA nanotechnology.

  11. Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells

    International Nuclear Information System (INIS)

    Mamo, Tewodros; Mladek, Ann C.; Shogren, Kris L.; Gustafson, Carl; Gupta, Shiv K.; Riester, Scott M.; Maran, Avudaiappan; Galindo, Mario; Wijnen, Andre J. van; Sarkaria, Jann N.; Yaszemski, Michael J.

    2017-01-01

    Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PK CS ), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PK CS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PK CS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PK CS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PK CS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. - Highlights: • DNA-PKcs is consistently expressed in human osteosarcoma tissue and cell lines. • The DNA-PKcs inhibitor, KU60648, effectively radiosensitizes osteosarcoma cells. • Combining KU60648 with radiation increases G2/M accumulation and DNA damage.

  12. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  13. A Role of hIPI3 in DNA Replication Licensing in Human Cells.

    Science.gov (United States)

    Huang, Yining; Amin, Aftab; Qin, Yan; Wang, Ziyi; Jiang, Huadong; Liang, Lu; Shi, Linjing; Liang, Chun

    2016-01-01

    The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

  14. Radiometric study of the incorporation of labelled precursors of DNA, RNA and proteins in cell cultures inoculated with Herpes simplex virus

    International Nuclear Information System (INIS)

    Dundarov, S.; Todorov, S.; Kavaklova, L.; Tsilka, E.; Rangelova, S.; Dundarova, D.

    1976-01-01

    Results are reported of a comparative investigation of tritium-labelled thymidine, uridine and leucine incorporation in cells inoculated with HSV-1 and HSV-2. There were no essential differences in the dynamics of the biosynthesis processes in either type of herpes viruses. Viral infection led to a marked activation of DNA synthesis and progressive inhibition of overall RNA synthesis. The rate of labelled amino-acid incorporation remained almost unchanged. There was a gradual increase in 3 H-thymidine incorporation, reaching a minimum by the 12th-16th hour and a rapid decrese thereafter. After the 12th hour part of the thymidine already incorporated in DNA (the thymidine pool) starts leaving the cells as a result of increased cell membrane permeability. The rate of changes in the biosynthesis processes in HSV-inoculated cells depends on infection multiplicity. Even in high inoculation multiplicity the blockade of cell biosynthesis is incomplete which furnishes a better opportunity for HSV reproduction. (author)

  15. Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

    Science.gov (United States)

    Hocek, Michal

    2014-11-07

    The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

  16. Toxicity of the bacteriophage λ cII gene product to Escherichia coli arises from inhibition of host cell DNA replication

    International Nuclear Information System (INIS)

    Kedzierska, Barbara; Glinkowska, Monika; Iwanicki, Adam; Obuchowski, Michal; Sojka, Piotr; Thomas, Mark S.; Wegrzyn, Grzegorz

    2003-01-01

    The bacteriophage λ cII gene codes for a transcriptional activator protein which is a crucial regulator at the stage of the 'lysis-versus-lysogeny' decision during phage development. The CII protein is highly toxic to the host, Escherichia coli, when overproduced. However, the molecular mechanism of this toxicity is not known. Here we demonstrate that DNA synthesis, but not total RNA synthesis, is strongly inhibited in cII-overexpressing E. coli cells. The toxicity was also observed when the transcriptional stimulator activity of CII was abolished either by a point mutation in the cII gene or by a point mutation, rpoA341, in the gene coding for the RNA polymerase α subunit. Moreover, inhibition of cell growth, caused by both wild-type and mutant CII proteins in either rpoA + or rpoA341 hosts, could be relieved by overexpression of the E. coli dnaB and dnaC genes. In vitro replication of an oriC-based plasmid DNA was somewhat impaired by the presence of the CII, and several CII-resistant E. coli strains contain mutations near dnaC. We conclude that the DNA replication machinery may be a target for the toxic activity of CII

  17. Relationship between DNA replication and DNA repair in human lymphocytes proliferating in vitro in the presence and in absence of mutagen

    International Nuclear Information System (INIS)

    Szyfter, K.; Wielgosz, M.Sz.; Kujawski, M.; Jaloszynski, P.; Zajaczek, S.

    1995-01-01

    The effects of mutagens on DNA replication and DNA repair were studied in peripheral blood lymphocytes (PBL) obtained from 21 healthy subjects, 2 samples from healthy heterozygote of ''Xeroderma pigmentosum'' (XP) and 2 samples from patient with clinically recognised XP. Inter-individual variations were found in DNA replication and in the level of spontaneous DNA repair measured under standard culture condition. Exposure of human PBL proliferating in vitro to B(a)P was followed by a partial inhibition of replicative DNA synthesis in all subjects and by an induction of DNA repair in healthy subjects. In XP patients DNA repair synthesis remained at the level attributed to spontaneous DNA repair. The response to mutagen varied individually. Results were analysed statistically. It was established that the studied indices of DNA synthesis correlate well with each other. The highest correlation was found between the levels of spontaneous and B(a)P-induced DNA repair. It is concluded that the level of spontaneous DNA repair is predictive for an estimation of cells ability to repair DNA damage. Inter-individual variations in the inhibition of DNA replication and in DNA repair synthesis are also dependent on the type of mutagen as shown by effects of other mutagens. Different effects of mutagen exposure on the inhibition of DNA replicative synthesis and induction of DNA repair can be explained by genetically controlled differences in the activity of enzymes responsible for mutagen processing and lesion removal. (author). 37 refs, 2 figs, 2 tabs

  18. Boron neutron capture therapy. Synthesis of boronated amines- and DNA intercalating agents for potential use in cancer therapy

    International Nuclear Information System (INIS)

    Ghaneolhosseini, H.

    1998-01-01

    Boron Neutron Capture Therapy is a binary cancer treatment modality, involving the delivery of a suitable boron compound to tumour cells followed by irradiation of the tumour by thermal neutrons. Boronated agents can selectively be delivered to tumour cells either directly with tumour-specific boron compounds, or by use of targeting strategies. However, the efficacy of this method would increase if the boron agents are localised in the cell nucleus rather than in the cell cytoplasm when neutron irradiation takes place. With these considerations in mind, some boronated DNA intercalating/interacting agents such as phenanthridine- acridine- spermidine- and naphthalimide derivatives were synthesised. Aminoalkyl-o-carboranes were synthesised in order to be used both for coupling to macromolecules and also for halogenation of their corresponding nido-derivatives. The amino groups were introduced using the Gabriel reagent N, N-dibenzyl iminodicarboxylate to provide 1-(aminomethyl)- and 1-(2-aminoethyl)-o-carboranes. The first attempt to achieve the possibility to accumulate a higher concentration of boron atoms in the cell nucleus was to synthesize carboranyl phenanthridinium analogues by reacting a p- or o-carboranyl moiety with phenanthridine, a chromophore with a planar aromatic ring system as DNA intercalator. Boronated acridine-spermidine, boronated diacridine, and boronated dispermidine were obtained in order to increase water solubility to avoid the interaction of these agents with non-DNA sides of the cell, especially membranes; and to enhance the feasibility of a higher DNA-binding constant and also decrease the DNA-drug dissociation rate. Finally, the synthesis of a boronated naphthalimide derivative was carried out by nucleophilic reaction of a primary aminoalkyl-p-carborane with naphthalic anhydride. Biological evaluations on DNA-binding, toxicity, and cellular binding with carboranyl phenanthridinium analogues, boronated acridine- and spermidine are described

  19. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels

    International Nuclear Information System (INIS)

    Cotten, M.; Laengle-Rouault, F.; Kirlappos, H.; Wagner, E.; Mechtler, K.; Zenke, M.; Beug, H.; Birnstiel, M.L.

    1990-01-01

    The authors have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, they have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle. They demonstrate here that this procedure is useful for a human leukemic cell line. They enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, they show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks transferrinfection, as does incubation of the cells at 18 degree C

  20. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  1. Preirradiation of host (monkey) cells mitigates the effects of UV upon simian virus 40 DNA replication

    International Nuclear Information System (INIS)

    Scaria, A.; Edenberg, H.J.

    1987-01-01

    The authors examined the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV40 DNA synthesis in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m 2 to SV40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m 2 in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the preirradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication. (Auth.)

  2. Unexpected Hydration of a Triple Bond During DNA Synthesis

    DEFF Research Database (Denmark)

    Fatthalla, Maha I.; Pedersen, Erik B.

    2016-01-01

    acidic conditions, polarizes the triple bond in the intercalator and this makes hydration of the triple bond possible during the DNA synthesis and an oligonucleotide with 1-(indol-3-yl)-2-(pyren-1-yl)ethanone as the intercalator is formed. Insertion of the unhydrated and hydrated linker systems gave...

  3. RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress

    DEFF Research Database (Denmark)

    Bhowmick, Rahul; Minocherhomji, Sheroy; Hickson, Ian D

    2016-01-01

    Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis...... replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective...

  4. Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

    Science.gov (United States)

    Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

    2012-01-01

    Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

  5. Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M

    2000-01-01

    Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccha......Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which......, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference...... transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently...

  6. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

    Science.gov (United States)

    Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

    2016-08-31

    DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

  7. UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp

    NARCIS (Netherlands)

    Buma, A.G.J.; Van Hannen, E.J.; Veldhuis, M.; Gieskes, W.W.C.

    1996-01-01

    The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

  8. UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp.

    NARCIS (Netherlands)

    Buma, A.G.J.; van Hannen, E.J; Veldhuis, M.J W; Gieskes, W.W C

    The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

  9. Effect of γ-irradiated DNA on the activity of DNA polymerase

    International Nuclear Information System (INIS)

    Leadon, S.A.; Ward, J.F.

    1981-01-01

    A cell-free assay was developed to measure the effect of γ-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. Irradiated poly(dA-dT) was more effective in reducing polymerase activity than calf thymus DNA. Thus the polymerase-inhibition site(s) appears to be associated with base damage, specifically adenine or thymine. Using a free-radical scavenger, OH radicals were found to be involved in producing the damage sites. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme and not for other proteins present in the assay. The inhibition of DNA polymerase occurred prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation

  10. DNA methylation in human fibroblasts following DNA damage and repair

    International Nuclear Information System (INIS)

    Kastan, M.B.

    1984-01-01

    Methylation of deoxycytidine (dCyd) incorporated by DNA excision repair synthesis in human diploid fibroblasts following damage with ultraviolet radiation (UV), N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene was studied utilizing [6- 3 H]dCyd to label repaired DNA specifically and high performance liquid chromatographic analysis to quantify the percentage of deoxycytidine converted to 5-methyldeoxycytidine (m 5 dCyd). In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication a level of 3.4% m 5 dCyd is reached in less than 2 hours, following UV-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approx.2.0% m 5 dCyd in the repair patch. This undermethylation of repair patches occurs throughout the genome. In cells from cultures in logarithmic-phase growth, m 5 dCyd formation in UV-induced repair patches occurs faster and to a greater extent, reaching a level of approx.2.7% in 10-20 hours. Pre-existing hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites. The distribution within chromatin of m 5 dCyd in repair patches was also investigated. Over a wide range of extents of digestion by staphylococcal nuclease or deoxyribonuclease I, the level of hypomethylation in repaired DNA in nuclease sensitive and resistant regions of chromatin was constant relative to the genomic level of methylation in these regions. Similar conclusions were reached in experiments with isolated mononucleosomes

  11. DNA alkylation and tumor induction in regenerating rat liver after cell cycle-related continuous N-nitrosodimethylamine infusion

    Energy Technology Data Exchange (ETDEWEB)

    Rabes, H.M.; Kerler, R.; Wilhelm, R.

    1983-01-01

    Synchronized regenerating rat liver after partial hepatectomy was used to study cell cycle-related DNA base alkylation and liver carcinogenesis. A continuous iv infusion of (/sup 14/C)N-nitrosodimethylamine (DMN) at a dose of 0.5 mg/kg/hour was given to inbred male Wistar Af/Han rats over a period of 8 hours either during the G1 phase, hydroxyurea-synchronized DNA synthesis, or the G2+M-phase of regenerating liver or to untreated rats (G0-phase liver--carcinogen dose, 1.5 mg/kg/hour). Two hours after the end of the infusion, the amount of 7-methylguanine was highest in the G0-phase liver, with a decrease in the G1 phase, the S-phase, and the G2+M-phase. After continuous DMN exposure, the O/sub 6/-methylguanine:7-methylguanine ratio was lower in the S-phase and G2+M-phase livers than in the G0-phase and G1-phase livers, indicating an increased O/sub 6/-methylguanine repair during DNA synthesis and the G2+M-phase. Liver tumors in rats treated by continuous DMN infusion either during the G0 phase or the S-phase developed only after carcinogen exposure during DNA synthesis.

  12. Autoradiographic examinations on the synthesis of the virus herpes simplex in macrophages, rabbit kidney- and BHK-cells by labelling with 3H-thymidine, 3H-uridine and UDP-3H-galactose

    International Nuclear Information System (INIS)

    Lindermann, J.

    1979-01-01

    After injection, an autoradiographically measurable reduplication of virus-specific DNA takes place in NMRI-macrophages infected with HSV (herpes simplex virus). For this process, a sufficient duration of the 3 H-thymidine pulses is of essential importance. An infection with HSV of the Lennette strain induces in considerably more cells of a macrophage population the production of virus-specific DNA than the infection with HSV of the D-316 type. After injection a reduplication of virus-specific DNA occurs in more cells of a HSV-infected population of old macrophages than in that one consisting of young macrophages. In the 7th hour after injection a reduction of RNA synthesis was observed in the HSV-infected macrophage cultures. After HSV-infection, a synthesis of virus-specific DNA could not be detected autoradiographically in macrophages of C 57/bl mice. After HSV-infection the virus-specific DNA synthesis was also observed in activated macrophages of NMRI mice, which had been treated with thioglucollate 72 hours before cell extraction. In general, the previous thioglucollate treatment increased the susceptibility of the macrophages of infection with subsequent reduplication of herpes-specific DNA. Compared to other not infected but activated macrophages, the RNA synthesis after injection was found to be not (HSV Lennette) or only minutely (HSV D-316) reduced in HSV-infected cultures of activated macrophages. In the investigations about the utilisation of exogenic UDP- 3 H-galactose by BHK and rabbit cells it was observed that only few cells of the cultures provide an externally accessible galactosyl transferase activity. Due to the influence of cell-damaging mechanisms a cell proliferation is induced, which is accompanied by externally accessible galactosyl transferase activities in the cell cultures. (orig./MG) [de

  13. DNA cytoskeleton for stabilizing artificial cells.

    Science.gov (United States)

    Kurokawa, Chikako; Fujiwara, Kei; Morita, Masamune; Kawamata, Ibuki; Kawagishi, Yui; Sakai, Atsushi; Murayama, Yoshihiro; Nomura, Shin-Ichiro M; Murata, Satoshi; Takinoue, Masahiro; Yanagisawa, Miho

    2017-07-11

    Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.

  14. Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900 MHz radiofrequency fields

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yulong; Zong, Lin; Gao, Zhen [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China); Zhu, Shunxing [Laboratory Animal Center, Nantong University, Nantong, Jiangsu Province (China); Tong, Jian [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China); Cao, Yi, E-mail: yicao@suda.edu.cn [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China)

    2017-03-15

    Highlights: • Increased reactive oxygen species. • Decreased mitochondrial transcription Factor A and polymerase gamma. • Decreased mitochondrial transcripts (ND1 and 16S) and mtDNA copy number. • Increased 8-hydroxy-2′deoxyguanosine. • Decreased adenosine triphosphate. - Abstract: HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900 MHz radiofrequency fields (RF) at 120 μW/cm{sup 2} power intensity for 4 h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2′-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells.

  15. Molecular mechanism of radioadaptive response: A cross-adaptive response for enhanced repair of DNA damage in adapted cells

    International Nuclear Information System (INIS)

    Takaji Ikushima

    1997-01-01

    The radioadaptive response (RAR) has been attributed to the induction of a repair mechanism by low doses of ionizing radiation, but the molecular nature of the mechanism is not yet elucidated. We have characterized RAR in a series of experiments in cultured Chinese hamster V79 cells. A 4-h interval is required for the full expression of RAR, which decays with the progression of cell proliferation. Treatments with inhibitors of poly(ADP-ribose) polymerase, protein- or RNA synthesis, and protein kinase C suppress the RAR expression. The RAR cross-reacts on clastogenic lesions induced by other physical and chemical DNA-damaging agents. The presence of newly synthesised proteins has been detected during the expression period. Experiments performed using single-cell gel electrophoresis provided more direct evidence for a faster and enhaced DNA repair rate in adapted cells. Here, using single-cell gel electrophoresis, a cross-adaptive response has been demonstrated for enhanced repair of DNA damage induced by neocarzinostatin in radio-adapted cells. (author)

  16. DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

    International Nuclear Information System (INIS)

    Ben-Ishai, R.; Sharon, R.; Rothman, M.; Miskin, R.

    1984-01-01

    We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s)

  17. Intestinal DNA concentration and protein synthesis in response to ...

    African Journals Online (AJOL)

    Performance, protein synthesis and mucosal DNA in small intestine of Leghorn hens may be affected by low quality feedstuff. An experiment was conducted in completely randomized design (CRD) in 2 × 2 factorial arrangement. Main factors included diets containing 20 and 40 % barley and black and blue strains of leghorn ...

  18. Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C

    International Nuclear Information System (INIS)

    Mullenders, L.H.F.; Kesteren, A.C. van; Bussmann, C.J.M.; Zeeland, A.A. van; Natarajan, A.T.

    1984-01-01

    The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimer-specific endonuclease V of bacteriophage T 4 . The results suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts the authors observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in regions of the genome. (Auth.)

  19. Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

    International Nuclear Information System (INIS)

    Rainbow, A.J.

    1989-01-01

    The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D 0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus. (author)

  20. Comparison of the effects of nafenopin, methyl clofenapate, WY-14,643 and clofibric acid on peroxisome proliferation and replicative DNA synthesis in rat liver

    International Nuclear Information System (INIS)

    Price, R.J.; Evans, J.G.; Lake, B.G.; Gangolli, S.D.

    1991-01-01

    A wide variety of chemicals have been shown to produce hepatic peroxisome proliferation (PP) in the rat and certain of these compounds are also hepatocarcinogens. In this study the authors have investigated the relationship between PP and cell replication in the rat liver. Male Sprague-Dawley rats were fed control diet or diet containing either 0.0125 and 0.05% nafenopin (NAF), 0.05% methyl clofenapate (MC), 0.025% Wy-14,643 (WY) or 0.5% clofibric acid (CA) for 1 and 15 wk. All four compounds produced marked liver enlargement and a sustained induction of peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidizing enzyme activities. Enzyme induction was less marked with 0.0125% NAF than with 0.05% NAF which was similar to that produced by the other three compounds. Replicative DNA synthesis was studied by implanting 7 day Alzet osmotic pumps containing [ 3 H]thymidine during wk 0-1 and 14-15. After 1 wk replicative DNA synthesis (assessed as radioactivity incorporated into homogenate DNA by scintillation counting) was increased in all treatment groups to 170-325% of control levels. Hepatocyte Labelling Index (determined by autoradiography of liver sections) was increased in all treated groups. After 15 wk hepatic DNA radioactivity levels were 155 and 200% of control in MC and WY treated rats, respectively, whereas NAF and CA had no effect. These results demonstrate that the relationship between the magnitude of PP and induction of cell replication depends on the compound being studied and that some peroxisome proliferators produce sustained stimulation of replicative DNA synthesis in the rat

  1. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  2. The effect of low radiation doses on DNA repair processes

    International Nuclear Information System (INIS)

    Tuschl, H.

    1978-08-01

    Error free DNA repair processes are an important preprequisite for the maintenance of genetic integrity of cells. They are of special importance for persons therapeutically or occupationally exposed to radiation. Therefore the effect of radiation therapy and elevated natural background radiation on unscheduled DNA synthesis was tested in peripheral lymphocytes of exposed persons. Both, autoradiographic studies of unscheduled DNA synthesis and measurement of 3 H-thymidine uptake into double stranded and single-strand containing DNA fractions revealed an increase of capacity for DNA repair. (author)

  3. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  4. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    Science.gov (United States)

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts of the template

    Energy Technology Data Exchange (ETDEWEB)

    Lockhart, M.L.; Deutsch, J.F.; Yamaura, I.; Cavalieri, L.F.; Rosenberg, B.H.

    1982-01-01

    The effects of DNA lesions produced by the carcinogenic alkylating agents ethylnitrosourea and diethylsulfate on the extent of DNA synthesis have been studied in a system utilizing circular single-stranded phi X174 DNA as template and a 392-base restriction fragment as primer with E. coli polymerase I (Klenow fragment). Apurinic sites produced by loss of unstable ethylated bases from the template terminate DNA synthesis at the first such site encountered, but ethyl adducts at most, if not all, locations permit readthrough. 22 references, 3 figures, 1 table.

  6. Nuclear Lipid Microdomain as Place of Interaction between Sphingomyelin and DNA during Liver Regeneration

    Directory of Open Access Journals (Sweden)

    Samuela Cataldi

    2013-03-01

    Full Text Available Nuclear sphingomyelin is a key molecule for cell proliferation. This molecule is organized with cholesterol and proteins to form specific lipid microdomains bound to the inner nuclear membrane where RNA is synthesized. Here, we have reported the ability of the sphingomyelin present in the nuclear microdomain to bind DNA and regulate its synthesis, and to highlight its role in cell proliferation induced by partial hepatectomy. During G1/S transition of the cell cycle, sphingomyelin and DNA content is very high and it is strongly reduced after exogenous sphingomyelinase treatment. During the S-phase of the cell cycle, the stimulation of sphingomyelinase and inhibition of sphingomyelin–synthase are accompanied by the DNA synthesis start. To assess the specificity of the results, experiments were repeated with trifluoperazine, a drug known to affect the synthesis of lipids and DNA and to stimulate sphingomyelinase activity. The activity of sphingomyelinase is stimulated in the first hour after hepatectomy and sphingomyelin–DNA synthesis is strongly attenuated. It may be hypothesized that the nuclear microdomain represents a specific area of the inner nuclear membrane that acts as an active site of chromatin anchorage thanks to the stabilizing action of sphingomyelin. Thus, sphingomyelin metabolism in nuclear lipid microdomains is suggested to regulate cell proliferation.

  7. Repair and cell-cycle response in cells exposed to environmental biohazards. Comprehensive project report, June 1, 1979-May 31, 1982

    International Nuclear Information System (INIS)

    Billen, D.

    1982-01-01

    Agents which cause damage to DNA leading to inhibition of DNA synthesis or faulty DNA replication or repair may cause cell death or mutation. Many organisms possess the ability to circumvent some or all of this DNA damage. Many DNA mutants of E. coli and B. subtilis provide a genetic approach to measuring the role of individual components of the DNA repair and replicative system. The information obtained with prokaryotes provides leads to assess the details of DNA repair and replication in mammalian systems including man. Escherichia coli cells treated with a low concentration of toluene become permeable to a variety of compounds, including the precursors and cofactors necessary for DNA synthesis. By their manipulation various aspects of DNA replication and repair can be selectively emphasized. Observations made by use of this system include: (1) Repair synthesis induced by x irradiation or exposure to alkylating chemicals of toluene-treated cells is more extensive if polynucleotide ligase is inhibited. (2) DNA replication in E. coli is carried out by DNA polymerase III. The replication of DNA is strongly inhibited by methylmethansulfonate, N-methyl-N-nitrosourea, and N-methyl-N'-nitro-N-nitrosoguanidine. (3) Using a po1A1 po1B100 dnaB (po1I - , po1II - , po1III + ) mutant of E. coli, it was demonstrated that the dnaB gene product is not necessary for Po1III directed repair synthesis. (4) The physiological stage of cells and tissues affects their response to environmental hazards. (5) Procedures for permeabilizing mammalian cells have been developed or further refined; and (6) In earlier studies involving both alkylating agents and x rays, it was observed that the number of DNA single-strand breaks increased with dose along with repair synthesis. It appears that non-repaired sites do not serve as primer ends for Po1I-dependent repair synthesis in toluene-treated cells

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

    Science.gov (United States)

    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.

  9. Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

    Energy Technology Data Exchange (ETDEWEB)

    Cornelis, J.; Su, Z.Z.; Dinsart, C.; Rommelaere, J. (Universite libre de Bruxelles, Rhode St Genese (Belgium))

    The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.

  10. Nucleolar TRF2 attenuated nucleolus stress-induced HCC cell-cycle arrest by altering rRNA synthesis.

    Science.gov (United States)

    Yuan, Fuwen; Xu, Chenzhong; Li, Guodong; Tong, Tanjun

    2018-05-03

    The nucleolus is an important organelle that is responsible for the biogenesis of ribosome RNA (rRNA) and ribosomal subunits assembly. It is also deemed to be the center of metabolic control, considering the critical role of ribosomes in protein translation. Perturbations of rRNA synthesis are closely related to cell proliferation and tumor progression. Telomeric repeat-binding factor 2 (TRF2) is a member of shelterin complex that is responsible for telomere DNA protection. Interestingly, it was recently reported to localize in the nucleolus of human cells in a cell-cycle-dependent manner, while the underlying mechanism and its role on the nucleolus remained unclear. In this study, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1), a nucleolar protein that is responsible for the nucleolus construction and rRNA synthesis, interacted with TRF2 and mediated the shuttle of TRF2 between the nucleolus and nucleus. Abating the expression of NOLC1 decreased the nucleolar-resident TRF2. Besides, the nucleolar TRF2 could bind rDNA and promoted rRNA transcription. Furthermore, in hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC7721, TRF2 overexpression participated in the nucleolus stress-induced rRNA inhibition and cell-cycle arrest.

  11. Autoradiography of DNA from Hela cells under normal conditions and after treatment with hydroxyurea

    International Nuclear Information System (INIS)

    Martinova, Y.S.; Angelova, P.A.; Roeva, I.G.

    1984-01-01

    The results are presented of the first stage of the elaboration of the novel autoradiographic technique for studying the replication of DNA fibers from nonsynchronized Hela cell cultures under normal conditions and after treatment with hydroxyurea. The preparations were covered with liquid nuclear emulsion Ilford L 4 . Exposure was carried out for 3 months at 4 deg C. After development, the autoradiograms were recorded quantitatively, and the length of the individual replicative segments was measured by means of an object micrometers. For each group (control and experimental) 100 segments from different cells were recorded. The results obtained were subjected to mathematical-statistical processing for determining the standard deviation. The application of hidroxyurea highly reduces the replicative elements, i.e. it actually inhibits DNA synthesis. This inhibition is due to reduction in the production of the four endogenous deoxynucleotides and affects the length of growth of the DNA chain, but the interreplicative distance as well

  12. Comparison of the effect of L-lysine-. cap alpha. -oxidase from Trichoderma harzianum Rifai and Trichoderma viride on nucleic acid synthesis in human tumor cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Khaduev, S.K.; Zhukova, O.S.; Dobrynin, Ya.V.; Soda, K.; Berezov, T.T.

    1986-10-01

    This paper gives comparative data on the effect of the new antitumor enzyme LO from a Soviet strain Trichoderma harzianum Rifai and from Trichoderma viride from Japan, on DNA and RNA synthesis in human ovarian carcinoma cells (CaOv strain) in culture, and also the results of the action of LO from T. harzianum Rifai on protein synthesis. Specific precursors were added before the end of the incubation time to the samples; /sup 3/H-thymidine as precursor for DNA synthesis, /sup 3/H-uridine as RNA precursor, and /sup 3/H-leucine as protein precursor. The final values of inhibition of DNA and RNA synthesis in the presence of enzyme from both sources are shown to depend in a virtually linear manner on enzyme concentration.

  13. Determination of radioinduced delay in DNA synthesis in two-garlic-clones cells (Allium Sativum L.)

    International Nuclear Information System (INIS)

    Perez Lezcano, A.; Perez Talavera, S.

    1989-01-01

    To contribute to tech improvement of the use of ionizing radiations as an auxiliary tool in the fitoimprovement, dose-effect curves for the 'Martinez' and 'Sancti Spiritus-3' clones were stablished by using as effect the delay induced by radiations in DNA synthesis determined by the 'Martinez' clone which induces a delay of 50% in reference to the control is approximately 11 Gy, while the dose value for the 'Sancti Spiritus-3' clone is 18 Gy, thus the 'Martinez' clones has a higher sensitivity to radiations than the other clone, therefore it coincides with what we found for these clones other indexes are used as radiosensitivity criteria

  14. DNA repair , cell repair and radiosensitivity

    International Nuclear Information System (INIS)

    Zhestyanikov, V.D.

    1983-01-01

    Data obtained in laboratory of radiation cytology and literature data testifying to a considerable role of DNA repair in cell sensitivity to radiation and chemical DNA-tropic agents have been considered. Data pointing to the probability of contribution of inducible repair of DNA into plant cells sensitivity to X-rays are obtained. Certain violations of DNA repair do not result in the increase of radiosensitivity. It is assumed that in the cases unknown mechanisms of DNA repair operate

  15. DNA to DNA transcription might exist in eukaryotic cells

    OpenAIRE

    Li, Gao-De

    2016-01-01

    Till now, in biological sciences, the term, transcription, mainly refers to DNA to RNA transcription. But our recently published experimental findings obtained from Plasmodium falciparum strongly suggest the existence of DNA to DNA transcription in the genome of eukaryotic cells, which could shed some light on the functions of certain noncoding DNA in the human and other eukaryotic genomes.

  16. Engineered cell-cell communication via DNA messaging

    Directory of Open Access Journals (Sweden)

    Ortiz Monica E

    2012-09-01

    Full Text Available Abstract Background Evolution has selected for organisms that benefit from genetically encoded cell-cell communication. Engineers have begun to repurpose elements of natural communication systems to realize programmed pattern formation and coordinate other population-level behaviors. However, existing engineered systems rely on system-specific small molecules to send molecular messages among cells. Thus, the information transmission capacity of current engineered biological communication systems is physically limited by specific biomolecules that are capable of sending only a single message, typically “regulate transcription.” Results We have engineered a cell-cell communication platform using bacteriophage M13 gene products to autonomously package and deliver heterologous DNA messages of varying lengths and encoded functions. We demonstrate the decoupling of messages from a common communication channel via the autonomous transmission of various arbitrary genetic messages. Further, we increase the range of engineered DNA messaging across semisolid media by linking message transmission or receipt to active cellular chemotaxis. Conclusions We demonstrate decoupling of a communication channel from message transmission within engineered biological systems via the autonomous targeted transduction of user-specified heterologous DNA messages. We also demonstrate that bacteriophage M13 particle production and message transduction occurs among chemotactic bacteria. We use chemotaxis to improve the range of DNA messaging, increasing both transmission distance and communication bit rates relative to existing small molecule-based communication systems. We postulate that integration of different engineered cell-cell communication platforms will allow for more complex spatial programming of dynamic cellular consortia.

  17. Effect of mode of administration of methyl methanesulfonate and triethylenemelamine on induction of unscheduled DNA synthesis in mouse germ cells

    International Nuclear Information System (INIS)

    Sheu, C.W.; Sega, G.A.; Owens, J.G.

    1987-01-01

    The effect of route of administration on induction of unscheduled DNA synthesis (UDS) in mouse germ cells in vivo was studied using two germ cell mutagens, methyl methanesulfonate (MMS) and triethylenemelamine (TEM). The chemicals were administered to male mice (C3Hf x 101)F 1 by IP injection or gavage using acute or 5-day subacute regimens. After completion of dosing, methyl-[ 3 H]thymidine ([ 3 H]TdR) was injected into the testes, and spermatozoa were collected 16 days later. The sperm heads were isolated, and UDS was determined by the amount of [ 3 H]TdR incorporated. Acute administration of MMS (2-100 mg/kg) induced a strong, dose-related UDS response. The response was slightly higher with IP injection than with gavage. Acute administration of TEM (0.05-4.0 mg/kg) by IP injection or gavage induced weak and variable responses. The study showed that gavage, as well as IP injection, can be used for the administration of test chemicals and that the subacute 5-day regimen induced a higher UDS response than the acute regimen. Furthermore, the testicular route may enhance the detection of weak UDS inducers

  18. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40 0 C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G 1 -phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35 0 C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs

  19. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    Energy Technology Data Exchange (ETDEWEB)

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

  20. Physiological effects in bovine lymphocytes of inhibiting polyamine synthesis with ethylglyoxal bis(guanylhydrazone).

    Science.gov (United States)

    Igarashi, K; Morris, D R

    1984-11-01

    Previous results have suggested that ethylglyoxal bis(guanylhydrazone) is a more specific inhibitor of polyamine biosynthesis than the widely used methylglyoxal bis(guanylhydrazone). The physiological effects on mitogenically activated lymphocytes of polyamine depletion with ethylglyoxal bis(guanylhydrazone) were examined. In the presence of ethylglyoxal bis(guanylhydrazone) and the ornithine decarboxylase inhibitor alpha-difluoromethylornithine, the cellular contents of putrescine, spermidine, and spermine were decreased by 75 to 90, 65 to 80, and 40 to 60%, respectively, compared with control cultures. Inhibition of DNA synthesis in these polyamine-deficient cells was always greater than that of protein synthesis. Upon addition of spermidine to the deficient cells, the cellular spermidine content was restored within 4 hr, but the complete recovery of macromolecular synthesis took 10 to 20 hr. Thymidine kinase and DNA polymerase alpha activities in polyamine-deficient cells were lower than those in normal cells, whereas RNA polymerase II and leucyl transfer RNA synthase activities were nearly equal to those in normal cells. These results and studies with 2-dimensional gel electrophoresis raise the possibility that polyamines may regulate the synthesis of specific proteins. Decreased synthesis of replication proteins in polyamine-deficient cells may be one reason for the reduced synthesis of DNA.

  1. DNA repair in murine embryonic stem cells and differentiated cells

    International Nuclear Information System (INIS)

    Tichy, Elisia D.; Stambrook, Peter J.

    2008-01-01

    Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells

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

    International Nuclear Information System (INIS)

    Hochmann, J.; Stambergova, H.

    1988-01-01

    Histoautoradiography was used to detect dimethylnitrosamine-induced 3 H-thymidine incorporation in vivo into G phase hepatocytes. The description of the standard procedure for counting the grains and the mode of mathematical evaluation are 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 detecting hydroxyurea-resistant incorporation of 3 H-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 of striking differences between the experimental and the control groups a qualitative conclusion may be drawn even without counting the grains. (author). 5 tabs., 10 refs

  3. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

    Directory of Open Access Journals (Sweden)

    Elisa Mentegari

    2016-08-01

    Full Text Available DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

  4. Cell-specific monitoring of protein synthesis in vivo.

    Directory of Open Access Journals (Sweden)

    Nikos Kourtis

    Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

  5. DNA polymerase iota (Pol ι) promotes invasion and metastasis of esophageal squamous cell carcinoma.

    Science.gov (United States)

    Zou, Shitao; Shang, Zeng-Fu; Liu, Biao; Zhang, Shuyu; Wu, Jinchang; Huang, Min; Ding, Wei-Qun; Zhou, Jundong

    2016-05-31

    DNA polymerase iota (Pol ι) is an error-prone DNA polymerase involved in translesion DNA synthesis (TLS) that contributes to the accumulation of DNA mutations. We recently showed that Pol ι is overexpressed in human esophageal squamous cell cancer (ESCC) tissues which promotes ESCC' progression. The present study was aimed at investigating the molecular mechanisms by which Pol ι enhances the invasiveness and metastasis of ESCC cells. We found that the expression of Pol ι is significantly higher in ESCCs with lymph node metastasis compared to those without lymph node metastasis. Kaplan-Meier analysis revealed an inverse correlation between Pol ι expression and patient prognosis. The expression levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), two essential regulators of cells' invasiveness, were positively associated with Pol ι expression in ESCC tissues. Ectopic expression of Pol ι enhanced the motility and invasiveness of ESCC cells as evaluated by wound-healing and transwell assays, respectively. A xenograft nude mouse model showed that Pol ι promotes the colonization of ESCC cells in the liver, lung and kidney. Signaling pathway analysis identified the JNK-AP-1 cascade as a mediator of the Pol ι-induced increase in the expression of MMP-2/9 and enhancement of ESCC progression. These data demonstrate the underlying mechanism by which Pol ι promotes ESCC progression, suggesting that Pol ι is a potential novel prognostic biomarker and therapeutic target for ESCC.

  6. Cellular Responses to Cisplatin-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Alakananda Basu

    2010-01-01

    Full Text Available Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.

  7. The role of hRev7, the accessory subunit of hPolζ, in translesion synthesis past DNA damage induced by benzo[a]pyrene diol epoxide (BPDE

    Directory of Open Access Journals (Sweden)

    Maher Veronica M

    2010-12-01

    Full Text Available Abstract Background DNA polymerase zeta (Polζ is a specialized DNA polymerase that, unlike classical replicative polymerases, is capable of replicating past DNA lesions, i.e. of performing translesion synthesis (TLS. The catalytic subunit of hPolζ, hRev3, has been shown to play a critical role in DNA damage-induced mutagenesis in human cells, but less is known about the role of hRev7, the accessory subunit of hPolζ, in such mutagenesis. To address this question, we recently generated human fibroblasts with very significantly reduced levels of hRev7 protein and demonstrated that hRev7 is required to protect cells from ultraviolet(254 nm (UV radiation-induced cytotoxicity and mutagenesis (McNally et al., DNA Repair 7 (2008 597-604. The goal of the present study was to determine whether hRev7 is similarly involved in the tolerance of DNA damage induced by benzo[a]pyrene diol epoxide (BPDE, the reactive form of the widespread environmental carcinogen benzo[a]pyrene. Methods To determine whether hRev7 also plays a role in protecting human cells from the cytotoxicity and mutagenesis induced by benzo[a]pyrene diol epoxide (BPDE, cell strains with reduced hRev7 were compared to their parental strain and a vector control strain for the effect of BPDE on cell survival, induction of mutations, and the ability to progress through the cell cycle. Results The results show that cell strains with reduced hRev7 are more sensitive to the cytotoxic effect of BPDE than the control strains, and progress through S-phase at a slower rate than the control cells following BPDE treatment, indicating that hRev7, and likely hPolζ, is required for efficient bypass of BPDE-induced DNA lesions. However, neither the frequency nor kinds of mutations induced by BPDE in cells with reduced hRev7 differ significantly from those induced in the control strains, suggesting that hPolζ is not essential for inserting nucleotides opposite BPDE-induced DNA damage. Conclusions Taken

  8. Dynamic changes of peripheral blood T-lymphocyte DNA-Synthesis in rabbits after fractionated and single exposure to 60Co-γ rays

    International Nuclear Information System (INIS)

    Wang Zongwu; Chen Tiehe; Yu Zhijie; Han Ling; Pan Yusha; Su Fuqiang

    1988-01-01

    The experiments in 59 rabbits γ-irradiated with doses of 0, 0.5, 1.0, 2.0, and 3.0 Gy in fractional and single exposure to 60 Co-γ rays were reported, respectively · Dynamics of the changes of DNA-Synthesis in T-lymphocytes of peripheral blood was obserced during 29 days after γ-irradiation. Marked inhibition in DNA-synthesis was found on 1st day after irradiation. Recovery was observed in 3rd day after irradiation. The levels of DNA-synthesis before irradiation was recovered on 7th day after exposure for all groups. For fractionated irradiation, however, an increase, rather than a decrese, of DNA-synthesis was in the group of 1.0 Gy

  9. [Construction of fetal mesenchymal stem cell cDNA subtractive library].

    Science.gov (United States)

    Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

    2002-04-01

    To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

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

    Science.gov (United States)

    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.

  11. The background of mitochondrial DNA haplogroup J increases the sensitivity of Leber's hereditary optic neuropathy cells to 2,5-hexanedione toxicity.

    Directory of Open Access Journals (Sweden)

    Anna Ghelli

    Full Text Available Leber's hereditary optic neuropathy (LHON is a maternally inherited blinding disease due to mitochondrial DNA (mtDNA point mutations in complex I subunit genes, whose incomplete penetrance has been attributed to both genetic and environmental factors. Indeed, the mtDNA background defined as haplogroup J is known to increase the penetrance of the 11778/ND4 and 14484/ND6 mutations. Recently it was also documented that the professional exposure to n-hexane might act as an exogenous trigger for LHON. Therefore, we here investigate the effect of the n-hexane neurotoxic metabolite 2,5-hexanedione (2,5-HD on cell viability and mitochondrial function of different cell models (cybrids and fibroblasts carrying the LHON mutations on different mtDNA haplogroups. The viability of control and LHON cybrids and fibroblasts, whose mtDNAs were completely sequenced, was assessed using the MTT assay. Mitochondrial ATP synthesis rate driven by complex I substrates was determined with the luciferine/luciferase method. Incubation with 2,5-HD caused the maximal loss of viability in control and LHON cells. The toxic effect of this compound was similar in control cells irrespective of the mtDNA background. On the contrary, sensitivity to 2,5-HD induced cell death was greatly increased in LHON cells carrying the 11778/ND4 or the 14484/ND6 mutation on haplogroup J, whereas the 11778/ND4 mutation in association with haplogroups U and H significantly improved cell survival. The 11778/ND4 mutation on haplogroup U was also more resistant to inhibition of complex I dependent ATP synthesis by 2,5-HD. In conclusion, this study shows that mtDNA haplogroups modulate the response of LHON cells to 2,5-HD. In particular, haplogroup J makes cells more sensitive to its toxic effect. This is the first evidence that an mtDNA background plays a role by interacting with an environmental factor and that 2,5-HD may be a risk element for visual loss in LHON. This proof of principle has broad

  12. Radiation-induced apoptosis in F9 teratocarcinoma cells

    International Nuclear Information System (INIS)

    Langley, R.E.; Palayoor, S.T.; Coleman, C.N.; Bump, E.A.

    1994-01-01

    We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis. (Author)

  13. Radiation-induced apoptosis in F9 teratocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Langley, R E; Palayoor, S T; Coleman, C N; Bump, E A [Joint Center for Radiation Therapy and Dana Farber Cancer Inst., Boston (United States)

    1994-05-01

    We have found that F9 murine teratocarcinoma cells undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation. We studied the time course, radiation dose-response, and the effects of protein and RNA synthesis inhibitors on this process. The response is dose dependent in the range 2-12 Gy. Internucleosomal DNA fragmentation can be detected as early as 6 h postirradiation and is maximal by 48 h. Cycloheximide, a protein synthesis inhibitor, and 5,6-dichloro-1-[beta]-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, both induced internucleosomal DNA fragmentation in the unirradiated cells and enhanced radiation-induced DNA fragmentation. F9 cells can be induced to differentiate into cells resembling endoderm with retinoic acid. After irradiation, differentiated F9 cells exhibit less DNA fragmentation than stem cells. This indicates that ionizing radiation can induce apoptosis in non-lymphoid tumours. We suggest that embryonic tumour cells may be particularly susceptible to agents that induce apoptosis. (Author).

  14. Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA.

    Science.gov (United States)

    Richards, W L; Song, M K; Krutzsch, H; Evarts, R P; Marsden, E; Thorgeirsson, S S

    1985-07-01

    We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.

  15. Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test

    International Nuclear Information System (INIS)

    Raffin, A.L.

    2009-06-01

    DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

  16. NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

    Science.gov (United States)

    Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

    2015-01-01

    Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

  17. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

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

    DEFF Research Database (Denmark)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia

    2016-01-01

    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...

  19. Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12

    International Nuclear Information System (INIS)

    Billen, D.; Hellermann, G.R.

    1976-01-01

    Toluene-treated Escherichia coli mutants have been used to study the roles of deoxyribonucleic acid (DNA) polymerases I, II, and III, and of DNA ligase in repair synthesis and strand rejoining following X-irradiation. In cells possessing all three DNA polymerases, both a greater amount of repair synthesis (''exaggerated'' repair synthesis) and failure of ligation are observed when DNA ligase activity is inhibited. In a mutant lacking the polymerizing activity of DNA polymerase I, exaggerated repair synthesis is not observed, and strand rejoining does not occur even if DNA ligase is fully activated. In a mutant possessing the polymerizing activity of DNA polymerase I but lacking its 5' → 3' exonuclease activity, exaggerated repair synthesis is minimal. After irradiation, DNA polymerases II and III are capable of carrying out an adenosine 5'-triphosphate-dependent repair synthesis, but rejoining of strand breaks does not occur and exaggerated synthesis is not seen whether DNA ligase is active or not. These results suggest that DNA polymerase I and DNA ligase act together to limit repair synthesis after X irradiation and that both are necessary in toluene-treated cells for strand rejoining. DNA polymerases II and III apparently cannot complete chain elongation and gap filling, and therefore repair carried out by these enzymes does not respond to ligase action

  20. Comparison of radiation-induced DNA-protein cross-links formed in oxic, hypoxic, and glutathione depleted cells

    International Nuclear Information System (INIS)

    Xue, L.; Friedman, L.R.; Chiu, S.; Ramakrishnan, N.; Oleinick, N.L.

    1987-01-01

    Treatment of cells with L-buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH). Subsequent metabolism depletes the cells of GSH. GSH-depletion sensitizes both oxic and hypoxic cells to the lethal effects of ionizing radiation. DNA-protein cross-links (DPC) are formed preferentially between DNA sequences active in transcription and a subset of proteins of the nuclear matrix. Thus, DPC may be an indicator lesion of damage in sensitive regions of the genome. The interrelationships between GSH level, oxic vs. hypoxic status, and the yield of DPC have been studied in terms of number of lesions and repair rate in Chinese hamster V79 and in human lung carcinoma A549 cells. The data suggest that elevated background levels of DPC are indicative of a reduced repair capacity, and greater radiation-induced yields of DPC in hypoxia may also be indicative of a compromised repair mechanism

  1. Scheduled and unscheduled DNA synthesis in chick embryo liver following X-irradiation and treatment with DNA repair inhibitors in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Stammberger, I.; Tempel, K. (Muenchen Univ. (Germany, F.R.). Inst. fuer Pharmakologie und Toxikologie); Schmahl, W. (Gesellschaft fuer Strahlen- und Umweltforschung m.b.H. Muenchen, Neuherberg (Germany, F.R.). Inst. fuer Pathologie)

    1989-09-01

    Three hours following X-irradiation of chick embryos with doses of 4 and 8 Gy the in vitro incorporation of tritiated thymidine (({sup 3}H)dT) into DNA (scheduled DNA synthesis, ss) of hepatocytes was reduced to about one-third. Within 24 h after the exposure, ss returned to control values. The return of ss to a normal rate could be strongly inhibited by 2', 3'-dideoxythymidine (ddT), and to a lesser extent by 1-beta-D-arabinofuranosylcytosine (araC). In strong contrast to ss, the hydroxyurea (hu)-resistant ({sup 3)H}dT incorporation (unscheduled DNA synthesis, us) showed a highly significant increase 24 h after treatment of the embryos with araC and/or X-irradiation. Autoradiographic studies revealed no change of total ({sup 3}H)dT labelling frequency in the whole chick embryo liver 24 h after treatment with araC and/or X-irradiation, but a persistent depression of ss and a simultaneous increase of us. (author).

  2. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

    International Nuclear Information System (INIS)

    Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

    1986-01-01

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells

  3. Tenofovir alafenamide (TAF) does not deplete mitochondrial DNA in human T-cell lines at intracellular concentrations exceeding clinically relevant drug exposures.

    Science.gov (United States)

    Stray, Kirsten M; Park, Yeojin; Babusis, Darius; Callebaut, Christian; Cihlar, Tomas; Ray, Adrian S; Perron, Michel

    2017-04-01

    HIV-infected patients treated with certain nucleoside reverse transcriptase inhibitors (NRTIs) have experienced adverse effects due to drug-related mitochondrial toxicity. Tenofovir alafenamide (TAF) is a novel prodrug of the NRTI tenofovir (TFV) with an improved safety profile compared to tenofovir disoproxil fumarate (TDF). Prior in vitro studies have demonstrated that the parent nucleotide TFV has no significant effects on mtDNA synthesis. This study investigated whether clinically relevant TAF and TDF exposures affect mtDNA content in human lymphocytes. First, activated or resting peripheral blood mononuclear cells (PBMCs), as well as MT-2 and Jurkat T-cell lines, were continuously treated with ddC for 10 days to establish their susceptibility to mtDNA depletion. PBMCs had low sensitivity to NRTI-mediated mtDNA depletion in vitro. In contrast, ddC treatment of rapidly dividing MT-2 and Jurkat cells resulted in a dose-dependent decrease in mtDNA. Therefore, these two T-cell lines were selected for evaluating TAF and TDF treatment effects. MT-2 and Jurkat cells were pulse-treated with TAF or TDF every 24 h for 10 days to mimic pharmacologically relevant drug exposures. Pulse treatment of cells with 3.3 μM TAF or 1.1 μM TDF for 10 days resulted in 2- to 7-fold greater steady-state intracellular TFV-diphosphate (TFV-DP) levels than those observed clinically in TAF- or TDF-treated patients. At these concentrations, no significant TAF- (106.7% and 84.1% of control; p = 0.77 and 0.12 for MT-2 and Jurkat, respectively) or TDF- (100.6% and 91.0% of control; p = 0.91 and 0.37, respectively) associated reduction in mtDNA content was observed compared with untreated control cells. This study demonstrates that, despite delivering higher intracellular levels of TFV-DP than TDF, TAF does not inhibit mtDNA synthesis in vitro at concentrations exceeding the clinically relevant intracellular drug exposures. Thus, TAF has a low potential for mitochondrial toxicity in

  4. The DNA topoisomerase II catalytic inhibitor merbarone is genotoxic and induces endoreduplication

    International Nuclear Information System (INIS)

    Pastor, Nuria; Domínguez, Inmaculada; Orta, Manuel Luís; Campanella, Claudia; Mateos, Santiago; Cortés, Felipe

    2012-01-01

    In the last years a number of reports have shown that the so-called topoisomerase II (topo II) catalytic inhibitors are able to induce DNA and chromosome damage, an unexpected result taking into account that they do not stabilize topo II-DNA cleavable complexes, a feature of topo II poisons such as etoposide and amsacrine. Merbarone inhibits the catalytic activity of topo II by blocking DNA cleavage by the enzyme. While it was first reported that merbarone does not induce genotoxic effects in mammalian cells, this has been challenged by reports showing that the topo II inhibitor induces efficiently chromosome and DNA damage, and the question as to a possible behavior as a topo II poison has been put forward. Given these contradictory results, and the as yet incomplete knowledge of the molecular mechanism of action of merbarone, in the present study we have tried to further characterize the mechanism of action of merbarone on cell proliferation, cell cycle, as well as chromosome and DNA damage in cultured CHO cells. Merbarone was cytotoxic as well as genotoxic, inhibited topo II catalytic activity, and induced endoreduplication. We have also shown that merbarone-induced DNA damage depends upon ongoing DNA synthesis. Supporting this, inhibition of DNA synthesis causes reduction of DNA damage and increased cell survival.

  5. Modifications of DNA clamps and their role in DNA damage management

    NARCIS (Netherlands)

    Wit, N.

    2013-01-01

    We show that proliferating cell nuclear antigen (PCNA) modification plays an important role in the efficient regulation of mammalian translesion DNA synthesis (TLS), but, as opposed to lower eukaryotes such as yeast, TLS polymerases can also be activated without ubiquitinated PCNA (PCNA-Ub). Future

  6. Achieving Precision Death with Cell-Cycle Inhibitors that Target DNA Replication and Repair.

    Science.gov (United States)

    Lin, Aimee Bence; McNeely, Samuel C; Beckmann, Richard P

    2017-07-01

    All cancers are characterized by defects in the systems that ensure strict control of the cell cycle in normal tissues. The consequent excess tissue growth can be countered by drugs that halt cell division, and, indeed, the majority of chemotherapeutics developed during the last century work by disrupting processes essential for the cell cycle, particularly DNA synthesis, DNA replication, and chromatid segregation. In certain contexts, the efficacy of these classes of drugs can be impressive, but because they indiscriminately block the cell cycle of all actively dividing cells, their side effects severely constrain the dose and duration with which they can be administered, allowing both normal and malignant cells to escape complete growth arrest. Recent progress in understanding how cancers lose control of the cell cycle, coupled with comprehensive genomic profiling of human tumor biopsies, has shown that many cancers have mutations affecting various regulators and checkpoints that impinge on the core cell-cycle machinery. These defects introduce unique vulnerabilities that can be exploited by a next generation of drugs that promise improved therapeutic windows in patients whose tumors bear particular genomic aberrations, permitting increased dose intensity and efficacy. These developments, coupled with the success of new drugs targeting cell-cycle regulators, have led to a resurgence of interest in cell-cycle inhibitors. This review in particular focuses on the newer strategies that may facilitate better therapeutic targeting of drugs that inhibit the various components that safeguard the fidelity of the fundamental processes of DNA replication and repair. Clin Cancer Res; 23(13); 3232-40. ©2017 AACR . ©2017 American Association for Cancer Research.

  7. Chromosomal DNA replication of Vicia faba cells

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1976-01-01

    The chromosomal DNA replication of higher plant cells has been investigated by DNA fiber autoradiography. The nuclear DNA fibers of Vicia root meristematic cells are organized into many tandem arrays of replication units or replicons which exist as clusters with respect to replication. DNA is replicated bidirectionally from the initiation points at the average rate of 0.15 μm/min at 20 0 C, and the average interinitiation interval is about 16 μm. The manner of chromosomal DNA replication in this higher plant is similar to that found in other eukaryotic cells at a subchromosomal level. (auth.)

  8. Regulation of DNA repair processes in mammalian cell

    International Nuclear Information System (INIS)

    Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

    1992-01-01

    A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

  9. DNA-Conjugated Organic Chromophores in DNA Stacking Interactions

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V.; Pedersen, Erik Bjerregaard

    2009-01-01

    Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic ch...... review presents those efforts in the design of intercalators/organic chromophores as oligonucleotide conjugates that form a foundation for the generation of novel nucleic acid architectures......Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic...

  10. Synthesis and DNA cleavage activity of Bis-3-chloropiperidines as alkylating agents.

    Science.gov (United States)

    Zuravka, Ivonne; Roesmann, Rolf; Sosic, Alice; Wende, Wolfgang; Pingoud, Alfred; Gatto, Barbara; Göttlich, Richard

    2014-09-01

    Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Role of DNA polymerase α in chromosomal aberration production by ionizing radiation

    International Nuclear Information System (INIS)

    Bender, M.A.

    1983-01-01

    Aphidicolin is a tetracyclic diterpinoid fungal antibiotic which inhibits DNA synthesis in eukaryotic cells by interfering specifically with DNA polymerase α, apparently by binding to and inactivating the DNA-polymerase α complex. We have shown that aphidicolin, like other inhibitors of DNA synthesis, both induces chromosomal aberrations in human peripheral lymphocytes, and, as a post-treatment, interacts synergistically with x rays to produce greatly enhanced aberration yields. The present experiments explore the effects of aphidicolin in human lymphocytes in the post-DNA-synthetic G 2 phase of the cell cycle. These experiments utilized labeling with tritiated thymidine to positively identify cells in the S phase at the time of treatment, and used serial colcemid collections and fixations to determine aberration yields over as much of the G 2 phase as feasible. Because DNA polymerase α is the only DNA synthetic or repair enzyme known to be affected by aphidicolin, we infer that this enzyme is directly involved in the repair of DNA lesions which can result in visible chromosomal aberrations. (DT)

  12. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

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

    International Nuclear Information System (INIS)

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

    1991-01-01

    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

  14. DNA-repair and mutations in immuncompetent cells from patients with rheumatic diseases and corresponding animal models

    International Nuclear Information System (INIS)

    Altmann, H.

    1977-01-01

    Unscheduled DNA synthesis was investigated in lymphocytes of patients with different inflammatory rheumatic diseases. After γ-irradiation H 3 -thymidin incorporation in DNA and DNA rejoining was reduced. After UV-irradiation the first step (90 min) of unscheduled DNA synthesis was above the controls. Some animal models for human diseases showed the same trend. An infectious ethiology was discussed for some of these diseases. (author)

  15. DNA-specific labelling by deoxyribonucleoside 5'-monophosphates in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Brendel, M.; Faeth, W.W.; Toper, R.

    1975-01-01

    Growth of 5'-dTMP low-requiring strains is inhibited by exogenous 5'-dGMP and 5'-GMP at concentrations higher than 5 x 10 -4 M. Synthesis of nucleic acids ceases and cells remain fixed in their respective place in the cell cycle. At concentrations lower than 10 -5 M deoxyribonucleoside 5'-monophosphates may be employed for radioactive labelling, the label being preferentially used for DNA synthesis. Affinity to DNA of the 5'-dNMPs is in the order of 5'-dAMPS > 5'-dGMP > 5'-dCMP > 5'-dUMP. DNA-specific label is achieved with 5'-dAMP when the medium is supplemented with adenine and deoxyadenosine. (orig.) [de

  16. The influence of inhibitors on dimer removal and repair of single-strand breaks in normal and bromodeoxyuridine substituted DNA of HeLa cells

    International Nuclear Information System (INIS)

    Cornelis, J.J.

    1978-01-01

    The elimination of cyclobutane pyrimidine dimers from the nuclear DNA of ultraviolet irradiated HeLa cells has been examined by means of chromatography and immunoautoradiography. The extent and duration of the process was similar when dimers were assayed by both methods, proving that the antisera recognized pyrimidine dimers. The rate of dimer excision did not differ through the cell cycle with the exception of mitosis during which no dimers were removed. Dimer excision is a relatively fast process which is terminated within a few hours, but it leaves many dimers in the DNA. Excision is depressed by inhibitors of semiconservative DNA synthesis that affect the DNA precursor pool or DNA polymerases. Cells whose DNA is partly substituted with bromodeoxyuridine instead of thymidine, repair single-strand breaks and remove dimers at the same rate but to different extents. On the other hand, inhibitors limit repair of breaks and removal of dimers to the same degree suggesting that the repair of the two types of lesion is coordinated. (Auth.)

  17. The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery.

    Science.gov (United States)

    O'Reilly, Rachel K; Turberfield, Andrew J; Wilks, Thomas R

    2017-10-17

    Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures. The largest libraries made by conventional synthesis are currently of the order of 10 8 distinct molecules. To put this in context, there are 10 13 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a

  18. In vivo effects of T-2 mycotoxin on synthesis of proteins and DNA in rat tissues

    International Nuclear Information System (INIS)

    Thompson, W.L.; Wannemacher, R.W. Jr.

    1990-01-01

    Rats were given an ip injection of T-2 mycotoxin (T-2), the T-2 metabolite, T-2 tetraol (tetraol), or cycloheximide. Serum, liver, heart, kidney, spleen, muscle, and intestine were collected at 3, 6, and 9 hr postinjection after a 2-hr pulse at each time with [14C]leucine and [3H]thymidine. Protein and DNA synthesis levels in rats were determined by dual-label counting of the acid-precipitable fraction of tissue homogenates. Rats given a lethal dose of T-2, tetraol, or cycloheximide died between 14 and 20 hr. Maximum inhibition of protein synthesis at the earliest time period was observed in additional rats given the same lethal dose of the three treatments and continued for the duration of the study (9 hr). With sublethal doses of T-2 or tetraol, the same early decrease in protein synthesis was observed but, in most of the tissues, recovery was seen with time. In the T-2-treated rats. DNA synthesis in the six tissues studied was also suppressed, although to a lesser degree. With sublethal doses, complete recovery of DNA synthesis took place in four of the six tissues by 9 hr after toxin exposure. The appearance of newly translated serum proteins did not occur in the animals treated with T-2 mycotoxin or cycloheximide, as evidenced by total and PCA-soluble serum levels of labeled leucine. An increase in tissue-pool levels of free leucine and thymidine in response to T-2 mycotoxin was also noted. T-2 mycotoxin, its metabolite, T-2 tetraol, and cycloheximide cause a rapid inhibition of protein and DNA synthesis in all tissue types studied. These results are compared with the responses seen in in vitro studies

  19. Aging and photo-aging DNA repair phenotype of skin cells-Evidence toward an effect of chronic sun-exposure

    Energy Technology Data Exchange (ETDEWEB)

    Prunier, Chloe; Masson-Genteuil, Gwenaeelle [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France); Ugolin, Nicolas [Laboratoire de Cancerologie Experimentale, CEA, DSV, IRCM, SREIT, BP6, Fontenay-aux-Roses Cedex F-92265 (France); Sarrazy, Fanny [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France); Sauvaigo, Sylvie, E-mail: sylvie.sauvaigo@cea.fr [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France)

    2012-08-01

    Several studies have demonstrated the deleterious effect of aging on the capacity of cells to repair their DNA. However, current existing assays aimed at measuring DNA repair address only a specific repair step dedicated to the correction of a specific DNA lesion type. Consequently they provide no information regarding the repair pathways that handle other types of lesions. In addition to aging, consequences of photo-exposure on these repair processes remain elusive. In this study we evaluated the consequence of aging and of chronic and/or acute photo-exposure on DNA repair in human skin fibroblasts using a multiplexed approach, which provided detailed information on several repair pathways at the same time. The resulting data were analyzed with adapted statistics/bioinformatics tools. We showed that, irrespective of the repair pathway considered, excision/synthesis was less efficient in non-exposed cells from elderly compared to cells from young adults and that photo-exposure disrupted this very clear pattern. Moreover, it was evidenced that chronic sun-exposure induced changes in DNA repair properties. Finally, the identification of a specific signature at the level of the NER pathway in cells repeatedly exposed to sun revealed a cumulative effect of UVB exposure and chronic sun irradiation. The uses of bioinformatics tools in this study was essential to fully take advantage of the large sum of data obtained with our multiplexed DNA repair assay and unravel the effects of environmental exposure on DNA repair pathways.

  20. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

    Science.gov (United States)

    Seco, Elena M.

    2017-01-01

    Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

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