Electron transfer to 5-bromouracil from their nucleobase electron adducts and their protonated forms has been studied by product analysis. When an electron is transferred to 5-bromouracil, the ensuing 5-bromouracil radical anion rapidly loses a bromide ion. The uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as a function of (N)/(5-BrU) after γ-radiolysis of Ar-saturated solutions it is concluded that the hetero atom protonated forms transfer electron quantitatively to 5-bromouracil. (author). 3 refs., 1 fig
Bromouracil mutagenesis was studied in several strains of E. coli in combination with measurement of incorporation of bromouracil in DNA. For levels below 10% total replacement of bromouracil for thymine, mutagenesis was negligible compared with higher levels of incorporation. Such a nonlinear response occurred both when the bromouracil was evenly distributed over the genome and when a small proportion of the genome was highly substituted. Also, the mutation frequency could be drastically lowered by amino acid starvation following bromouracil incorporation. These observations suggest the involvement of repair phenomena. Studies of mutagenesis in recA/sup -/ and uvrA/sup -/ mutants, as well as studies of prophage induction, did not support an ''error prone'' repair pathway of mutagenesis. On the other hand, uvrD/sup -/ and uvrE/sup -/ mutants, which are deficient in DNA mismatch repair, had much increased mutation frequencies compared with wild type cells. The mutagenic action of bromouracil showed specificity under the conditions used, as demonstrated by the inability of bromouracil to revert an ochre codon that was easily revertable by ultraviolet light irradiation. The results are consistent with a mechanism of bromouracil mutagenesis involvng mispairing, but suggest that the final mutation frequencies depend on repair that removes mismatched bases.
SAWANT M. S.; ZHANG Shicui; WANG Qingyin
Zebrafish (Danio rerio) genetic screens allow isolation of a wide array of problems in vertebrate biology. The effects of base analogues 5-bromouracil and 6-aminopurine on the development of zebrafish embryos are reported for the first time in this study. The early development of the zebrafish embryos was little affected by 5-bromouracil and 6-aminopurine, while the late development (organogenesis) was significantly impaired. Embryos exposed to 5-bromouracil mainly showed curled tail, wavy body, golden pigmentation and the mouth with protruding lower jaw. 6-aminopurine-treated embryos had defective anterior structures, curled tails and wavy body. RAPD analysis showed that the majority of 5-bromouracil- and 6-aminopurine-treated larvae and fish shared banding patterns in common with the control, suggesting that most mutagenesis induced by these agents are point mutations. However, some fish derived from 5-bromouracil-treated embryos had golden (gol) pigmentation; and RAPD analysis revealed that their band patterns differed from those of the control.Possibly, 5-bromouracil can occasionally cause relatively extensive changes in the fish genome. The results of this study may provide valuable help for detailed studies of mutagenesis.
Abdoul-Carime, H.; Huels, M. A.; Brüning, F.; Illenberger, E.; Sanche, L.
We report measurements of dissociative electron attachment (DEA) to gaseous 5-bromouracil (BrU) for incident electron energies between 0 and 16 eV. Low energy electron impact on BrU leads not only to the formation of a long lived parent anion BrU-, but also various anion fragments resulting from endo- and exo-cyclic bond ruptures, such as Br-, uracil-yl anions, i.e., (U-yl)-, OCN-, and a 68 amu anion tentatively attributed to H2C3NO-. The incident electron energy dependent signatures of either the Br- and (U-yl)- yields (at 0, 1.4, and 6 eV), or the OCN- and H2C3NO- yields (at 1.6 and 5.0 eV) suggests competing DEA channels for anion fragment formation. The production cross sections, at 0 eV incident electron energy, for BrU-, Br-, and (U-yl)- are estimated to be about 6×10-15, 6×10-14, and 1.0×10-15 cm2, respectively.
The fragmentation of singly and multiply charged 5-bromouracil molecules (C4H3N2O2Br) induced by collisions with slow multiply charged ions has been studied. The emission of neutral fragments as well as charge separating decay channels are identified as a function of the projectile charge state. In the first case, delayed loss of neutral moieties, occurring on a μs time scale, indicates a wider internal energy distribution resulting in a power law decay. In the second case, the most important decay channels, leading to the formation of Br+, HNCO+ and CO+/NHCH+, are discussed showing that in many processes intramolecular H-migration occurs before fragmentation. Furthermore, molecular rearrangement may lead to delayed charge separating processes. Although the dication of bromouracil is unstable, smaller doubly charged systems created by the loss of neutral fragments are found to be (meta) stable. (authors)
Jacob, H.E. (Akademie der Wissenschaften der DDR, Jena. Zentralinstitut fuer Mikrobiologie und Experimentelle Therapie); Golovinsky, E. (Bylgarska Akademiya na Naukite, Sofia)
The presence of 5-bromouracil (BU) as well as 5-bromo-2-deoxyuridine (BUdR) in the cultivation media of bacteria results in the distinct increase of UV sensitivity. With the nucleic acid base analogue 8-azaadenine (8-AA) a similar effect was confirmed, however, not so pronounced. The combined action of BU or BUdR and 8-AA on Escherichia coli, Proteus mirabilis, Bacillus subtilis and Bacillus cereus was investigated. The sensitization effect of BUdR does not increase if 8-AA is present additionally during cultivation. On the contrary, a decrease of sensibilization occurs. This may be caused by the protective effect of the adenine derivative against UV irradiation, if it is present in the cell, but not incorporated into the DNA.
The presence of 5-bromouracil (BU) as well as 5-bromo-2-deoxyuridine (BUdR) in the cultivation media of bacteria results in the distinct increase of UV sensitivity. With the nucleic acid base analogue 8-azaadenine (8-AA) a similar effect was confirmed, however, not so pronounced. The combined action of BU or BUdR and 8-AA on Escherichia coli, Proteus mirabilis, Bacillus subtilis and Bacillus cereus was investigated. The sensitization effect of BUdR does not increase if 8-AA is present additionally during cultivation. On the contrary, a decrease of sensibilization occurs. This may be caused by the protective effect of the adenine derivative against UV irradiation, if it is present in the cell, but not incorporated into the DNA. (author)
Holroyd, Leo F; van Mourik, Tanja
The potential energy surfaces of stacked base pairs consisting of cytosine (C), thymine (T), uracil (U) and the mutagenic thymine analogue 5-bromouracil (BrU) have been searched to obtain all possible minima. Minima and transition states were optimised at the counterpoise-corrected M06-2X/6-31+G(d) level, both in the gas phase and in water, modelled by the polarizable continuum model. The stacked dimers studied are BrU/BrU, C/BrU, C/C, C/T, C/U, T/BrU and T/U. Both face-to-back and face-to-face structures were considered. Free energies were calculated at 298.15 K. Together with U/U, T/T and BrU/U results from previous work, these results complete the family consisting of every stacked dimer combination consisting of C, T, U and BrU. The results were used to assess the hypothesis suggested in the literature that BrU stacks stronger than T, which could stabilise the mispair formed by BrU and guanine. In the gas phase, structures of C/BrU, T/BrU and U/BrU with greater zero-point-corrected binding energies than C/T, T/T and U/T, respectively, were found, with differences in favour of BrU of 3.1 kcal mol(-1), 1.7 kcal mol(-1) and 0.5 kcal mol(-1), respectively. However, the structure of these dimers differed considerably from anything encountered in DNA. When only the dimers with the most "DNA-like" twist (±36°) were considered, C/BrU and T/BrU were still more strongly bound than C/T and T/T, by 0.5 kcal mol(-1) and 1.7 kcal mol(-1), respectively. However, when enthalpic and/or solvent contributions were taken into account, the stacking advantage of BrU was reversed in the gas phase and mostly nullified in water. Enhanced stacking therefore does not seem a plausible mechanism for the considerably greater ability of BrU-G mispairs over T-G mispairs to escape enzymatic repair. PMID:26507806
Three C-6 radiohalogenated uracil derivatives were prepared by non-isotopic halogen exchange reactions for evaluation as diagnostic radiopharmaceuticals. [6-36Cl]chlorouracil (radiochemical yield 77%, specific activity 5.66 MBq mmol-1) was prepared via calcium [36Cl]chloride exchange on 6-iodouracil, [6-82Br]bromouracil (27%, 68.4 MBq mmol-1) was prepared via ammonium [82Br]bromide exchange on 6-iodouracil and [6-123I]iodouracil (55.4%, 5.41 GBq mmol-1) was prepared via sodium [123I]iodide exchange on 6-chlorouracil. The specific activities and radiochemical yields were dependent upon the halide-ion concentration. (author)
王艳花; 李立; 卢运祥; 邹建卫
Ab initio and density functional calculations were employed to investigate the bonding patterns in the adenine-5-bromouracil(AT+)complexes.It is shown that the Br atom in 5-bromouracil(T+)is involved in bonding both with the hydrogen atom of the amino group of adenine(A)and with N7(A)(or N1(A)).With this motif,the Br atom interacts with a nucleophile(H)in a"head-on"fashion and an electrophile(N)in a"side-on"fashion,forming both hydrogen and halogen bonds.Electrostatic attraction between the Br atom in T+and N7(or N1)of adenine was found via the electrostatic potential analysis.The existence of A bond critical point is identified for the halogen bonds and the topological parameters at the bond critical point indicate the typical closed-shellinteractions in the pairs.Natural bond orbital analysis suggests that the charge transfer from the lone pair of the nitrogen atom of adenine is mainly directed to the C-Br antibonding orbital.Finally,halogen bonds in the T+AT+A tetrads were also explored.%利用从头算和密度泛函理论研究了腺嘌呤(A)-5-溴尿嘧啶复合物中(T+)中的键合模式.研究结果表明,T+中的Br原子同时与A分子中的氨基氢和氮原子存在弱的相互作用,在这种结合模式中,Br原子与亲核基团H正面结合,同时与来电基团N侧面结合,分别形成氢键和卤键.静电势分析发现:T+中的Br原子与A中的N7(或N1)是通过静电相互吸引的.Br与N原子之间的相互作用通过分了中的原子理论得以证实.关键点的拓扑参数显示卤键是闭壳层相互作用.自然键轨道分析说明,A中N原子上孤对电子的电荷主要转移到C-Br的反键轨道.另外在T+AT+A四面体结构中也发现了卤键.
Reductive dehalogenation of 5-bromouracil by aliphatic organic radicals ·CO2-, ·CH2OH, ·CH(CH3)OH, and ·CH(CH3)O- have been studied in oxygen free aqueous solutions in the presence of organic additives: formate, methanol or ethanol. For radicals production 60Co γ-radiolysis was employed and the yield of bromide was measured by means of ion chromatography. Both radical anions have reducing potential negative enough to transfer an electron to BrU producing bromide ion and U· radical. High yields of bromide have been measured increasing proportional to the concentration of the corresponding organic additives at a constant dose rate. This is characteristic for a chain process where regeneration of radical ions occurs by H-atom abstraction by U· radical from formate or ethanol. Results with the neutral radicals conformed earlier proposition that the reduction reaction of α-hydroxyalkyl radicals proceeds by the proton-coupled electron transfer mechanism (). Thus, while both ·CH2OH and ·CH(CH3)OH did not react with BrU in water/alcohol solutions, addition of bicarbonate and acetate in mmol dm-3 concentrations, pH 7, brought about chain debromination to occur in the case of ·CH(CH3)OH radical as reactant. Under the same conditions phosphate buffer, a base with higher bulk proton affinity, failed to have any influence. The results are taken as additional proofs for the specific complex formation of α-hydroxyalkyl radicals with suitable bases which enhances radicals' reduction potential in comparison with only water molecules as proton acceptors. Rate constants for the H-atom abstraction from ethanol and formate by U· radicals have been estimated to amount to about ≥85 and 1200 dm3 mol-1 s-1, respectively.
Holroyd, Leo Frederick; van Mourik, Tanja
The authors gratefully acknowledge EaStCHEM for computer time on the EaStCHEM Research Computing Facility. LFH is grateful to the Engineering and Physical Sciences Research Council for studentship support through the Doctoral Training Account scheme (grant code EP/K503162/1).
Electron migration in irradiated solutions of DNA was investigated using 5-bromouracil synthetically incorporated into oligonucleotides of defined base composition as a molecular indicator of electron interactions. Solvated electrons interact quantitatively with 5-bromouracil, leading to a highly reactive 5-yl radical which can abstract an adjacent hydrogen atom to yield uracil. Yields of uracil, or loss of 5-bromouracil, from irradiated oligonucleotide samples were measured using gas chromatography-mass spectrometric analysis of their trimethylsilylated acid hydrolysates. (author)
DNA of Bacillus subtilis strain UVSS 19-8 M, of high ultraviolet sensitivity, was isolated after cultivating in medium containing bromouracil. Isopycnic banding in CsCl shows an unusual pattern with four bands, including an extra one halfway between those for hybrid and for DNA fully substituted with bromouracil. DNA of this band, amounting to 15-25% of the total DNA mass in one preparation, was isolated and investigated. The characteristics found for this DNA, namely transforming ability, electron microscopic picture, behavior during heat denaturation and gentle shear are in agreement with a fourstranded DNA unit similar to one of the structures postulated by Holliday as intermediates during genetic recombination. The amount of this DNA when the cells were given 5J/m2 of 254 nm UV. UVSS 19-8 M from which this DNA has been isolated is shown to be defective for transformation and transfection, and can be regarded as rec-. (orig./MG)
Rabbani, S. R.; Edmonds, D. T.; Gosling, P.
Using nuclear quadrupole double-resonance techniques, nitrogen-14 and deuterium nuclear quadrupole coupling constants and asymmetry parameters have been measured in uracil, 5-bromouracil, cytosine, adenine, xanthine, hypoxanthine, their nucleosides, 2-aminopyrimidine, and benzimidazole. Zeeman studies and the detection of the simultaneous transitions of neighboring nuclei allowed in many cases a complete assignment of the observed spectral lines to particular 14N and 2D sites.
Kuemmerle, N; R. Ley; Masker, W
Excision repair of ultraviolet radiation-induced damage in a wild-type strain of Escherichia coli has been examined, using two methods for characterizing the resynthesis step of the repair process. Comparison of data obtained after both isopycnic analysis of repaired deoxyribonucleic acid and sedimentation velocity analysis of deoxyribonucleic acid after selective photolysis of bromouracil-containing repaired regions has shown that the repaired deoxyribonucleic acid molecules contain a semico...
The Technical Report summarizes the results of the synthesis and microPET animal scanning of several compounds labeled with positron-emitting isotopes in normal, neonatal and kainic acid treated (seizure induced) rats as potential PET tracers to image the process of neurogenesis using positron emission tomography (PET). The tracers tested were 3'-deoxy-3'-[F-18]fluorothymidine ([F-18]FLT) and 5'-benzoyl-FTL, 1-(2'-deoxy-2'-[F-18]fluoro-B-D-arabinofuranosyl)-5-bromouracil (FBAU) and 3',5'-dibenzoyl-FBAU, N-[F-18]fluoroacetyl-D-glucosamine (FLAG) and tetraacetyl-FLAG, and L-[1-C-11]leucine
Filikov, A.V.; Myasoedov, N.F. (AN SSSR, Moscow. Inst. Molekulyarnoj Genetiki)
A reaction mechanism is proposed for tritium labelling by the solid-state catalytic reductive dehalogenation (SCRD) method based on hydrogen spillover. A model system (palladium membrane with a layer of the original organic compound) is used for a kinetic study of the debromination of 5-bromouracil and the isotope exchange of ..cap alpha..-alanine at pressure of 0.07-20 kPa. A kinetic model is considered for the spillover stoppage due to the contamination of penetration centres by the reaction product. Other possible causes of the spillover stoppage are discussed. 6 refs.; 3 figs.
Full Text Available A comparative study concerning the effects of chloramphenicol (100 μg ml-1, actidione (10 μg ml-1, 5-bromouracil (190 μg ml-1, actinomycin D (30 μg ml-1 and DL-ethionine (800 μg ml-1 on the chloroplast fine structure, 14CO2 incorporation and the Hill reaction activity was the subject of the experiments presented in this paper. The experiments were conducted on bean seedlings under the conditions when chlorophyll accumulation was inhibited only partially. The results obtained indicate that chloromphenicol is responsible for the reduction of the number of grana per section of plastid and for the formation of numerous vesicles in the stroma. In the presence of actidione, actinomycin D or DL-ethionine the lamellae are poorly differentiated into .stroma and granum regions and there occur disturbances in the typical orientation of lamellae within chloroplasts. Only in the presence of 5-bromouracil the development of chloroplast structure resemble that in control plants. A comparison of the results obtained with those published earlier (Więckowski et al., 1974; Ficek and Więckowski, 1974 shows that such processes as assimilatory pigment accumulation, the rate of CO2 fixation, the Hill reaction activity, and the development of lamellar system are suppressed in a different extent by the inhibitors used.
Molecular mechanisms induced by the effects of ionizing radiation on nucleic acids: Free radicals in 5-halogenated uracil derivatives after reaction of radiolysis products of water in low-temperature glass
This thesis deals with the molecular mechanisms induced by the effects of ionizing radiation on the DNA. The study has been made for the main purpose of clarifying the possible role of the indirect effect, namely the attack of diffusible water radicals, in the process of radiosensitivity enhancement due to the incorporation of bromouracil instead of thymine into the DNA. The results of the experiments can be summarized by the statement that among the reactions studied in the low-temperature glasses, none revealed such a clear difference between uracil and thymine on the one hand, and the halogenated uracils on the other, that this difference could suffice to explain in terms of quality and quantity the observed in-vivo enhancement of radiosensitivity by halogenated uracils. This conclusion is in agreement with the results of radiobiological measurements on phagae and bacteria which in all cases revealed no or only very slight enhancement of the radiosensitivity in the indirect effect subsequent to bromouracil incorporation. (orig./AJ)
Mangner, Thomas J.
The Technical Report summarizes the results of the synthesis and microPET animal scanning of several compounds labeled with positron-emitting isotopes in normal, neonatal and kainic acid treated (seizure induced) rats as potential PET tracers to image the process of neurogenesis using positron emission tomography (PET). The tracers tested were 3'-deoxy-3'-[F-18]fluorothymidine ([F-18]FLT) and 5'-benzoyl-FTL, 1-(2'-deoxy-2'-[F-18]fluoro-B-D-arabinofuranosyl)-5-bromouracil (FBAU) and 3',5'-dibenzoyl-FBAU, N-[F-18]fluoroacetyl-D-glucosamine (FLAG) and tetraacetyl-FLAG, and L-[1-C-11]leucine.
Syntheses of [5-2H]-, [6-2H]-uracil and [5-2H]-, [6-2H]-cytosine were investigated. The catalytic reaction of uracil or cytosine with 2H2 gas in alkaline media gave rise to [6-2H]-compounds almost exclusively. On the other hand, the reaction of 5-bromouracil or 5-bromocytosine with 2H2 gas gave rise to a mixture of [5-2H]-, [6-2H]- and [5-2H, 6-2H]-compounds depending on the experimental conditions. By controlling the temperature, the pressure of 2H2 gas and the amount of catalyst, [5-2H]-uracil and [5-2H]-cytosine were obtained. The isotopic distribution in each product was measured by 1H NMR spectroscopy combined with an HPLC method. (author)
Excision repair of ultraviolet radiation-induced damage in a wild-type strain of Escherichia coli has been examined, using two methods for characterizing the resynthesis step of the repair process. Comparison of data obtained after both isopycnic analysis of repaired deoxyribonucleic acid and sedimentation velocity analysis of deoxyribonucleic acid after selective photolysis of bromouracil-containing repaired regions has shown that the repaired deoxyribonucleic acid molecules contain a semicontinuous distribution of sizes of repair tracts. Further analysis of our data suggests two major classes of repair patches, one about 20 to 40 nucleotides in length, and the other containing 1600 to 2000 nucleotides. Under the conditions employed, approximately 2 to 10% of the fully repaired regions are long repair patches
The bromouracil-photolysis technique has been used to estimate the sizes of the repaired regions in normal human and xeroderma pigmentosum (XP) cells irradiated by γ-rays aerobically or anoxically. After one and a half hours of incubation, single-strand breaks were repaired and the repaired regions were small - one to two BrUra residues -for cells irradiated aerobically or anoxically. After a 20-hour incubation, the repaired region in normal cells showed a component mimicking U.V.-repair. There were large patches (approximately 30 BrUra residues) in the approximate ratios of one per six chain breaks for aerobic irradiation and one per three chain breaks for anoxic irradiation. XP cells, however, only showed large patches at 20 hours if they had been irradiated aerobically. Such regions could not be detected in XP cells irradiated anoxically. These results indicate (1) that some part of ionizing damage mimics excision of U.V. damage in that the repair patches are large and the repair takes an appreciable time; (2) the types of such damage depend on whether the irradiation is done aerobically or anoxically; and (3) XP cells are defective in repairing a component of anoxic damage. (author)
The principal experimental results are determination of the changes in DNA base sequence resulting from forward mutations in the cI (repressor) gene of lambda phage induced by various agents. For phage irradiated with ultraviolet light and assayed in lightly irradiated host cells to induce Weigle mutagenesis (targeted mutagenesis), two-thirds of the mutations are transitions. Most transitions seem to arise at the sites of Py(6-4)Pyo photoproducts, not at the more widely studied cyclobutane pyrimidine dimers. The other mutations induced by ultraviolet are equally divided among transversions, frameshifts and double mutation events. The latter, two closely spaced base changes or a base change plus a frameshift, should rarely revert and may be the deletions induced by ultraviolet which have been previously reported. Unirradiated phage assayed in host cells heavily irradiated with ultraviolet light (nontargeted mutagenesis) show mainly frameshift mutations. These frameshifts may arise from low intracellular activity of DNA polymerase I when the enzyme binds to host DNA damaged by irradiation of the cells. Mismatch repair greatly reduced the numbers of mutations from bromouracil (which induces transitions by base mispairing) and acridines which induce frameshifts at runs of G.C base pairs). Only when mismatch repair activity is saturated by the number of improperly stacked bases in the DNA does a high level of mutagenesis occur
Radiation-induced electron migration along DNA is a mechanism by which randomly produced stochastic energy deposition events can lead to non-random types of damage along DNA manifested distal to the sites of the initial energy deposition. Radiation-induced electron migration in nucleic acids has been examined using oligonucleotides containing 5-bromouracil (5-BrU). Interaction of 5-BrU with solvated electrons results in release of bromide ions and formation of uracil-5-yl radicals. Monitoring either bromide ion release or uracil formation provides an opportunity to study electron migration processes in model nucleic acid systems. Using this approach we have discovered that electron migration along oligonucleotides is significantly influenced by the base sequence and strandedness. Migration along 7 base pairs in oligonucleotides containing guanine bases was observed for oligonucleotides irradiated in solution, which compares with mean migration distances of 6-10 bp for Escherichia coli DNA irradiated in solution and 5.5 bp for E. coli DNA irradiated in cells. Evidence also suggests that electron migration can occur preferentially in the 5' to 3' direction along a double-stranded oligonucleotide containing a region of purine bases adjacent to the 5-BrU moiety. Our continued efforts will provide information regarding the contribution of electron transfer along DNA to formation of locally multiply damaged sites created in DNA by exposure to ionizing radiation. (Author)
Kuemmerle, N.B.; Ley, R.D.; Masker, W.E. (Oak Ridge National Lab., TN (USA). Biology Div.)
The resynthesis step of the excision repair pathway has been examined in Escherichia coli K12 strains isogenic except for mutations in the uvrD cistron. Strains mutant at the uvrD3, uvrD101, uvrE156, and recLl52 loci exhibited slight but distinct differences in their response to ultraviolet radiation. The repair capacity of the uvrD101 mutant was given special attention. Repair resynthesis in this mutant was saturated at fluences greater than about 20 J/m/sup 2/. Isopycnic analysis of repaired deoxyribonucleic acid from this strain revealed a two-fold increase over its wild-type counterpart in the amount of repair replication performed after a dose of 15 J/m/sup 2/. Sedimentation velocity analysis of DNA after selective photolysis of bromouracil-containing repaired regions showed that the uvrD101 mutation exerted its primary effect on the long-patch component of excision repair. The uvrD101 mutant performed long-patch repair at a larger number of sites than the number repaired by this mode in the wild-type strain; these patches were more extensive in length than the long-patch component in wild-type.
The resynthesis step of the excision repair pathway has been examined in Escherichia coli K12 strains isogenic except for mutations in the uvrD cistron. Strains mutant at the uvrD3, uvrD101, uvrE156, and recLl52 loci exhibited slight but distinct differences in their response to ultraviolet radiation. The repair capacity of the uvrD101 mutant was given special attention. Repair resynthesis in this mutant was saturated at fluences greater than about 20 J/m2. Isopycnic analysis of repaired deoxyribonucleic acid from this strain revealed a two-fold increase over its wild-type counterpart in the amount of repair replication performed after a dose of 15 J/m2. Sedimentation velocity analysis of DNA after selective photolysis of bromouracil-containing repaired regions showed that the uvrD101 mutation exerted its primary effect on the long-patch component of excision repair. The uvrD101 mutant performed long-patch repair at a larger number of sites than the number repaired by this mode in the wild-type strain; these patches were more extensive in length than the long-patch component in wild-type. (orig.)
Full Text Available Abstract Background Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. Results An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb. Conclusions The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs
马文杰; 康欣梅; 张清媛
目的 探讨番茄提取物在乳腺癌动物模型中对肿瘤增殖、凋亡及血管生成的影响.方法 建立二甲基苯葸诱导的大鼠乳腺癌模型,给予高中低剂量番茄提取物灌胃,应用5-溴-2-脱氧尿嘧啶核苷标记法检测细胞增殖;原位末端标记法检测细胞凋亡;免疫组化法检测肿瘤微血管密度.结论 番茄组乳腺肿瘤的增殖指数和肿瘤微血管密度均低于对照组,且高剂量番茄组中乳腺肿瘤的凋亡指数较对照组升高,差异具有显著性(P<0.05).结论番茄提取物在乳腺癌中具有抗增殖、促凋亡和抗血管生成活性,具有潜在的抗肿瘤应用价值.%Objective To investigate the effects of tomato extraction on tumor proliferation, apoptosis and angiogenesis in breast cancer animal model. Methods DMBA-induced breast caner models in SD rats were set up and received low-dose, middle-dose and high-dose tomato extraction by gavage. Tumor cell proliferation was detected by bromouracil deoxyriboside labeling, cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling, and tumor microvascular density was detected by immunohistochemical method. Results The tumor proliferative index and microvascular density were decreased in tomato group compared with control, and the apoptotic index was increased in high-dose tomato group. The difference was statistically significant (P <0. 05). Conclusion The tomato extraction can inhibit tumor proliferation, promote apoptosis and decrease angiogenesis in breast cancer, and it has potential value for application in cancer treatment.