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1.
[3H] DNA fromEscherichia coli and [3H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [3H] thymidine and [3H] DNA treatment which shows that the activity of [3H] DNA is utilized during the S phase. After application of [3H] thymidine, only cell nuclei in S phase were labelled. After the application of [3H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [3H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [3H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [3H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA. 相似文献
2.
The interactions between Hg2+, Ce3+, and the mixuure of Ce3+ and Hg2+, and DNA from fish intestine in vitro were investigated by using absorption spectrum and fluorescence emission spectrum.
The ultraviolet absorption spectra indicated that the addition of Hg2+, Ce3+, and the mixture of Ce3+ and Hg2+ to DNA generated an obviously hypochromic effect. Meanwhile, the peak of DNA at 205.2 nm blue-shifted and at 258.2 nm red-shifted.
The size of the hypochromic effect and the peak shift of DNA by metal ion treatments was Hg2+>Hg2++Ce3+>Ce3+. The fluorescence emission spectra showed that with the addition of Hg2+, Ce3+, and the mixture of Ce3+ and Hg2+ the emission peak at about 416.2 nm of DNA did not obviously change, but the intensity reduced gradually and the sequence
was Hg2+>Hg2++Ce2+>Ce3+. Hg2+, Ce3+, and the mixture of Ce3+ and Hg2+ had 1.12, 0.19, and 0.41 binding sites to DNA, respectively; the fluorescence quenching of DNA caused by the metal ions all
attributed to static quenching. The binding constants (K
A
) of binding siees were 8.98×104 L/mol and 1.02×104 L/mol, 5.12×104 L/mol and 1.10×103 L/mol, 6.66×104 L/mol and 2.36×103 L/mol, respectively. The results showed that Ce3+ could relieve the destruction of Hg2+ on the DNA structure. 相似文献
3.
A direct comparison of [3H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [3H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [3H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [3H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [3H]thymidine incorporation and isotope dilution assays. 相似文献
4.
Summary Al3+, Fe3+, V2+ or Be2+ when added to shaken suspensions of Petunia hybrida pollen in 10% sucrose —0.01% H3BO3 induce a strong unscheduled DNA synthesis as measured by incorporation of 3H-thymidine into pollen DNA. The metal ions (added in most cases as the chloride) gave maximum effect at approximately 2 mM. Weaker reactions are given by Ca2+, Zn2+, Mn2+, Cd2+ and Cr3+ in decreasing order of effectiveness, while twelve other metal ions were shown to be ineffective or to give very low reaction. The unscheduled DNA synthesis induced by Al3+ was not altered by hydroxyurea, nicotinamide, caffeine or cycloheximide. It was markedly affected by the pH of the medium, the optimum pH being 5.0, where there could be a tendency for some base-binding of the metal (in contrast to phosphate binding) at the high Al3+ to DNA mole ratio used. It was considered that the DNA synthesis induced by the metal ions represents a repair synthesis. A DNA polymerase activity was detected in pollen extracts. It showed a preference for Mn2+ over Mg2+ and was estimated to have more than enough activity to account for the unscheduled DNA synthesis in pollen given by the most effective inducer, Al3+. 相似文献
5.
A mononuclear macrocyclic complex NiIIL3a (L3a = dianion of 2,3-dioxo-5,6:13,14-dibenzo-9,10-cyclohexyl-7,12-bis(methoxycarbonyl)-1,4,8,11-tetraazacyclotetradeca-7,11-diene), which shows high DNA cleavage activity in the presence of H2O2, was reported in our previous work. Considering that many systems for natural enzyme-mediated DNA cleavage contain two or more metal active sites, two new trinuclear complexes [Cu(NiL3a)2(dca)2]·2CH3OH (abbreviated as Cu(NiL3a)2) and [Ag(NiL3a)2(NO3)]·2CH3OH·0.5H2O (abbreviated as Ag(NiL3a)2) were synthesized in this work, where dca is the dicyanamide. The complexes were structurally characterized by single crystal X-ray analysis. The central Cu(II) or Ag(I) atom is linked to two [NiL3a] ligands by oxamido bridges forming a trinuclear structure. In Cu(NiL3a)2, the central Cu(II) ion is in an octahedral coordination geometry. Whereas in Ag(NiL3a)2, the central Ag(I) ion is in a rarely reported trigonal-prismatic coordination geometry. The DNA cleavage behavior of the complexes in the presence of H2O2 was studied in detail. Comparing with the NiL3a, the trinuclear complex Ag(NiL3a)2 nearly has no ability to cleave DNA, whereas Cu(NiL3a)2 is a much better DNA cleavage agent. Cu(NiL3a)2 can efficiently convert supercoiled DNA to nicked DNA with a rate constant of 0.074 ± 0.002 min−1 when 40 μM Cu(NiL3a)2 and 0.6 mM H2O2 are used. The cleavage mechanism between the complex Cu(NiL3a)2 and plasmid DNA is likely to involve singlet oxygen as reactive oxygen species. Circular dichroism (CD) and fluorescence spectroscopy indicate that both Cu(NiL3a)2 and NiL3a bind to DNA by a groove binding mode, and the binding between Cu(NiL3a)2 and DNA is much stronger than that between NiL3a and DNA. The present results may provide some information for the design of efficient multinuclear artificial nucleases. 相似文献
6.
Summary Chinese hamster cells (Cl : 1) were labelled with3H-thymidine or125Iododeoxyuridine for 18 h and after 3 h in non-radioactive medium they were stored at 0° C up to 6 h. The number of DNA strand breaks observed after the labelling period (37° C) or after treatment at 0° C was determined using the DNA-unwinding technique.125I-decays in DNA were significantly more efficient than3H-decays in introducing unrepairable DNA strand breaks during the labelling period. 32% of125I-induced and 3% of3H-induced DNA strand breaks were unrepaired after 21 h at 37° C. Comparison between the effects of125I- or 3H-disintegrations in DNA in three different ways shows 7–12 times more pronounced effects for125I-decays. For125I-labelled cells 3–4 DNA strand breaks were found per decay and the corresponding value for3H- labelled cells was 2. 相似文献
7.
Breakage of Parental DNA Strands in Haemophilus influenzae by 313 nm Radiation after Replication in the Presence of 5-Bromodeoxyuridine 总被引:3,自引:0,他引:3
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Kenneth L. Beattie 《Biophysical journal》1972,12(11):1573-1582
Haemophilus influenzae was labeled with thymidine-3H (dThd), then grown in the presence of 5-bromodeoxyuridine (BrdUrd), and then irradiated with 313 nm light (a wavelength that selectively photolyzes DNA containing 5-bromouracil [BrUra]). Irradiation with 313 nm light induced breaks in the 3H-labeled strands in cells grown with BrdUrd at a much higher frequency than in 14C-labeled DNA of cells not exposed to BrdUrd. Breakage of the 3H-labeled strands was about 0.6% as efficient as that of fully BrUra-substituted DNA. During growth in the presence of BrdUrd, susceptibility to 313 nm-induced breakage of the 3H-labeled DNA strands increased, reaching a maximum in about one generation, and it decreased to zero during subsequent growth for one generation in medium containing dThd instead of BrdUrd. Heat denaturation of DNA extracted from dThd-3H-labeled cells grown in the presence of BrdUrd eliminated 313 nm-induced breakage of the 3H-labeled strands. It is concluded that breakage of the 3H-labeled DNA strands resulted from reaction with photoproducts in the base-paired, BrUra-containing strands, rather than from photolysis of BrdUrd incorporated into parental strands. It may be possible to utilize the phenomenon of interstrand breakage in physical studies of DNA replication. 相似文献
8.
9.
In sterile cultures of free barley embryos, N-methyl-N-nitrosourea (MNU) caused a decrease in the size of both template [14C]-labeled DNA and of daughter [3H]DNA strands as determined in alkaline sucrose gradients, and inhibited the rate of [3H]thymidine incorporation. In addition, duplexes containing [3H]-daughter DNA analyzed in BND cellulose contained more single-stranded regions in MNU-treated embryos than in the corresponding control. Incubation of MNU-treated embryos in nutrient medium for up to 18 h after the [3H]-labeling permitted the recovery of small-sized daughter DNA to full-sized strands and led to the enhancement of double-strandedness of DNA duplexes containing [3H]-labeled strands. If [3H]-labeling had been carried out 8–10 h after the MNU treatment, the size of daughter DNA, the proportion of double-strandedness and the rate of thymidine uptake into DNA partially increased in comparison with rates observed when labeling had been done just after or 3 h after the MNU treatment, but these variables did not reach the values of the corresponding controls. 相似文献
10.
Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pKA of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 103 M?1 and 0.136 × 103 M?1, respectively, for native DNA and 7.48 × 103 M?1 and 0.938 × 103 M?1, respectively, for denatured DNA. The pKA value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms. 相似文献
11.
D. Gersanovski P. Colson C. Houssier E. Fredericq 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1985,824(4):313-323
At low ionic strength, Tb3+ binding strongly alters the secondary structure of DNA. Circular dichroism and electro-optical techniques are more sensitive than fluorescence to study these alterations in double-stranded DNA, at low Tb3+/DNA phosphate () ratios. Both techniques yield the following conclusion: as is increased, native and sonicated DNA undergo a transition from the B- to ψ-form, the latter being a compact structure characteristic of aggregated DNA. Our study of alkylated DNA establishes that the accessibility of N-7 guanine to Tb3+ is clearly required for structural alterations in an aggregated state to occur. The chelation of the phosphate group and of the N-7 guanine by Tb3+ simultaneously alters the geometry of the sugar-phosphate backbone and the stacking interaction between the bases in double-stranded DNA. 相似文献
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14.
The metabolic fate of 1-β-d -arabinofuranosyl-5-[(E)-2-bromovinyl]uracil (BV-araU) in herpes simplex virus type 1-infected cells was studied using tritium-labeled BV-araU. [3H]BV-araU was selectively taken-up by infected cells. Approximately 10% of the total uptake of [3H]BV-araU was recovered from the acid-insoluble fraction at any time post-infection. Both cellular uptake of [3H]BV-araU and its incorporation into the acid-insoluble fraction increased with increasing incubation time through 8 hr post-infection. Uptake of [3H]BV-araU and its incorporation into the acid-insoluble fraction also increased proportionally to the duration of exposure to [3H]BV-araU. An alkaline sucrose gradient sedimentation analysis revealed that the radioactive DNA obtained from cells pulse-labeled with [3H]BV-araU were small DNA fragments which remained at the top following a chasing period in isotope-free medium, whereas that pulse-labeled with [3H]thymidine was chased to a fraction of high molecular weight DNA. Nuclease P1 digestion reduced 99% of the [3H]BV-araU-labeled DNA extracted from infected cells to a low molecular weight. Following digestion of [3H]BV-araU-labeled DNA with micrococcal nuclease and spleen exonuclease, all of the radioactivity was recovered as [3H]BV-araU 3′-monophosphate. Thus, BV-araU strongly inhibits the elongation of viral DNA strands as demonstrated by the alkaline sucrose gradient sedimentation analysis, whereas at least a portion of the [3H]BV-araU is incorporated inside viral DNA strands in infected cells. 相似文献
15.
Sameer Mirza Bryan J. Katafiasz Rakesh Kumar Jun Wang Shakur Mohibi Smrati Jain Channabasavaiah Basavaraju Gurumurthy Tej K. Pandita Bhavana J. Dave Hamid Band Vimla Band 《Cell cycle (Georgetown, Tex.)》2012,11(22):4266-4274
Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3−/− cells. Notably, Ada3−/− cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G₁ or G₂ phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3fl/fl and Ada3−/− cells confirmed higher residual DNA damage in Ada3−/− cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability. 相似文献
16.
《Cell cycle (Georgetown, Tex.)》2013,12(22):4266-4274
Cell cycle regulation and DNA repair following damage are essential for maintaining genome integrity. DNA damage activates checkpoints in order to repair damaged DNA prior to exit to the next phase of cell cycle. Recently, we have shown the role of Ada3, a component of various histone acetyltransferase complexes, in cell cycle regulation, and loss of Ada3 results in mouse embryonic lethality. Here, we used adenovirus-Cre-mediated Ada3 deletion in Ada3fl/fl mouse embryonic fibroblasts (MEFs) to assess the role of Ada3 in DNA damage response following exposure to ionizing radiation (IR). We report that Ada3 depletion was associated with increased levels of phospho-ATM (pATM), γH2AX, phospho-53BP1 (p53BP1) and phospho-RAD51 (pRAD51) in untreated cells; however, radiation response was intact in Ada3?/? cells. Notably, Ada3?/? cells exhibited a significant delay in disappearance of DNA damage foci for several critical proteins involved in the DNA repair process. Significantly, loss of Ada3 led to enhanced chromosomal aberrations, such as chromosome breaks, fragments, deletions and translocations, which further increased upon DNA damage. Notably, the total numbers of aberrations were more clearly observed in S-phase, as compared with G? or G? phases of cell cycle with IR. Lastly, comparison of DNA damage in Ada3fl/fl and Ada3?/? cells confirmed higher residual DNA damage in Ada3?/? cells, underscoring a critical role of Ada3 in the DNA repair process. Taken together, these findings provide evidence for a novel role for Ada3 in maintenance of the DNA repair process and genomic stability. 相似文献
17.
Evidence is presented to demonstrate the presence of W chromosome-specific repetitive DNA sequences in the female White Leghorn chicken, Gallus g. domesticus, based on two different experimental approaches. First, 3H-labelled, female chicken DNA was hybridized with excess, unlabelled, mercurated, male DNA, and unhybridized single-stranded 3H-DNA (3H-SHU-DNA) was recovered by SH-Sepharose and hydroxyapatite column chromatography. Approximately 24% of the hybridizable 3H-SHU-DNA was female-specific and localized on the W chromosome. The second approach was to examine female-specific DNA fragments among the digests of chicken DNA with various restriction endonucleases. Among them, we found that digestion with XhoI produced two prominent female-specific bands of 0.60 kb (= kilobase pairs) and 1.1 kb. The 0.60 kb fragment was isolated and 3H-labelled by nick-translation. Female-specificity of the 3H-XhoI—0.60 kb DNA was judged to be at least 95% under the conditions of hybridization with membrane filter-bound DNA. Presence of amplified XhoI—0.60 kb DNA on the W chromosome seems to be limited to different lines of G. g. domesticus and no such repeat was detected in three species belonging to other genera in the order Galliformes and in three species belonging to other avian orders. 相似文献
18.
C B Lozzio 《Experimental cell research》1971,69(2):377-383
The uptake of 3H-uridine into RNA and of 3H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either 3H-thymidine or 3H-uridine at selected times, and analyzed in autoradiographs. Incorporation of 3H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10−3 M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of 3H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis. 相似文献
19.
Olga V. Kirsanova Alexander V. Sergeev Ivan S. Yasko 《Nucleosides, nucleotides & nucleic acids》2017,36(6):392-405
The incorporation of chemotherapeutic agent 6-thioguanine (SG) into DNA is a prerequisite for its cytotoxic action. This modification of DNA impedes the activity of enzymes involved in DNA repair and replication. Here, using hemimethylated DNA substrates we demonstrated that DNA methylation by Dnmt3a-CD is reduced if DNA is damaged by the incorporation of SG into one or two CpG sites separated by nine base pairs. An increase in the number of SG substitutions did not enhance the effect. Dnmt3a-CD binding to either of SG-containing DNA substrates was not distorted. Our results suggest that SG incorporation into DNA may influence epigenetic regulation via DNA methylation. 相似文献
20.
The pulse height spectra and the relative efficiencies of aqueous suspensions of [3H]DNA T4D bacteriophages, of [3H]DNA Escherichia coli bacteria, and of [32P]DNA T4D phages were measured and compared to the results of [3H]thymidine and [32P]orthophosphate solutions, respectively. In all of our measurements a scintillation mixture based upon Triton X-100/toluene (0.5 kg/1 liter) was used. We explain the different effects of the chemical quench (e.g., by CCl4) and of the absorption of β energy inside the specimen (e.g., phages and bacteria) on the pulse height spectra by means of Bethe's theory of electron stopping power. We measured also the dependence of the relative efficiency on the content of aqueous suspensions of [3H]DNA and [32P]DNA phages in the sample, and compared the results to the relative efficiencies of aqueous [3H]thymidine and [32P]orthophosphate solutions, respectively. 相似文献