共查询到14条相似文献,搜索用时 0 毫秒
1.
Polymerase Synthesis of DNAs Bearing Vinyl Groups in the Major Groove and their Cleavage by Restriction Endonucleases 下载免费PDF全文
Michaela Mačková Dr. Radek Pohl Prof. Dr. Michal Hocek 《Chembiochem : a European journal of chemical biology》2014,15(15):2306-2312
DNA molecules containing 5‐vinyluracil, 5‐vinylcytosine, or 7‐deaza‐7‐vinyladenine were prepared by polymerase incorporation of the corresponding vinyl‐modified 2′‐deoxyribonucleoside triphosphates, and the influence of the vinyl group in the major groove of DNA on the cleavage by diverse type II restriction endonucleases (REs) was studied. The presence of 5‐vinyluracil was tolerated by most of the REs, whereas only some REs were able to cleave sequences containing 7‐deaza‐7‐vinyladenine. The enzyme ScaI was found to cleave DNA containing 5‐vinylcytosine efficiently but not DNA containing the related 5‐ethynylcytosine. All other REs failed to cleave sequences containing any cytosine modifications. 相似文献
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《Chembiochem : a European journal of chemical biology》2003,4(7):553-553
The cover picture shows a chemical restriction method in which DNA double strands are cleaved at the position of the artificial base 8‐oxoguanine (Goxo). As as result of the mild cleavage conditions, the DNA fragments remain biologically active and can be used in gene cloning experiments. As an example, the lac Z′ gene was amplified by PCR with 8‐oxoguanine‐modified primers, restricted by treatment with ammonia, ligated into a plasmid vector, transformed in Escherichia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure, and allows the design of any 3′ overhang, independent of the sequence of the cloned DNA. Further information can be found in the article by Giese et. al. on pp. 610–614 相似文献
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Macíčková-Cahová H Pohl R Hocek M 《Chembiochem : a European journal of chemical biology》2011,12(3):431-438
A series of six pyrimidine-modified dNTPs--5-ethynyl-, 5-phenyl-, and 5-(3-nitrophenyl)deoxycitidine and -deoxyuridine triphosphates--were prepared and incorporated by primer extension with Vent (exo-)polymerase to specific DNA sequences within or next to the recognition sequences of selected restriction endonucleases. The cleavage of these pyrimidine-modified DNA sequences by 13 restriction enzymes was then studied. Whereas the presence of any modified C within the target sequence completely prevented any restriction cleavage, most enzymes tolerated the presence of 5-ethynylU and two of them even the presence of 5-phenyl- and 5-(3-nitrophenyl)U. Modifications outside the recognition sequence were tolerated except in the case of phenyl derivatives with the PvuII enzyme. 5-EthynylC was used for protection of the recognition sequence from cleavage in the presence of the second unmodified copy of the same sequence that was cleaved. 相似文献
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Meyer C Meyer D Bickle TA Giese B 《Chembiochem : a European journal of chemical biology》2003,4(7):610-614
Cleavage of DNA single and double strands at an 8-oxoguanine-containing nucleotide occurs in 90 % yield if the modified oligonucleotide is treated with NH(3) and O(2) at 60 degrees C. The mechanism of this oxidative cleavage reaction was studied, and the reaction was applied to the generation of single-stranded overhangs on PCR-amplified DNA that can be ligated. As an example, the lac Z' gene was amplified by PCR with 8-oxoguanine modified primers, restricted by ammonia treatment, ligated into a plasmid vector, transformed in Escherischia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure with restriction enzymes, and it allows the design of any 3'-overhang independent of the sequence of the cloned DNA. 相似文献
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Douglas D. Young Dr. Jeane M. Govan Mark O. Lively Prof. Alexander Deiters Prof. 《Chembiochem : a European journal of chemical biology》2009,10(10):1612-1616
Removal by the light : The photochemical regulation of restriction endonucleases, which are important enzymes in molecular biology, has been investigated. Photolabile protecting groups have been installed on DNA substrates and have been demonstrated to inhibit restriction endonuclease activity until removed by UV light irradiation. Interestingly, these groups do not appear to dramatically affect initial binding of the enzyme to the DNA substrate, but rather prevent recognition of the specific cleavage site.
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Polymerase Synthesis and Restriction Enzyme Cleavage of DNA Containing 7‐Substituted 7‐Deazaguanine Nucleobases 下载免费PDF全文
Michaela Mačková Soňa Boháčová Dr. Pavla Perlíková Dr. Lenka Poštová Slavětínská Prof. Dr. Michal Hocek 《Chembiochem : a European journal of chemical biology》2015,16(15):2225-2236
Previous studies of polymerase synthesis of base‐modified DNAs and their cleavage by restriction enzymes have mostly related only to 5‐substituted pyrimidine and 7‐substituted 7‐deazaadenine nucleotides. Here we report the synthesis of a series of 7‐substituted 7‐deazaguanine 2′‐deoxyribonucleoside 5′‐O‐triphosphates (dGRTPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dGRTPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7‐substituted 7‐deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage. 相似文献
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DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity
Tariq Fahmi Xiaoying Wang Dmitry D. Zhdanov Intisar Islam Eugene O. Apostolov Alena V. Savenka Alexei G. Basnakian 《International journal of molecular sciences》2020,21(22)
Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I. 相似文献
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Hitoshi Katada Makoto Komiyama Prof. 《Chembiochem : a European journal of chemical biology》2009,10(8):1279-1288
The final cut . Two types of artificial tools (artificial restriction DNA cutter and zinc finger nuclease) that cut double‐stranded DNA through hydrolysis of target phosphodiester linkages, have been recently developed. The chemical structures, preparation, properties, and typical applications of these two man‐made tools are reviewed.
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Dr. Aashrita Rajagopalan Dr. Markus Schober Anita Emmerstorfer Lucas Hammerer Anna Migglautsch Dr. Birgit Seisser Dr. Silvia. M. Glueck Dr. Frank Niehaus Dr. Juergen Eck Dr. Harald Pichler Prof. Dr. Karl Gruber Prof. Dr. Wolfgang Kroutil 《Chembiochem : a European journal of chemical biology》2013,14(18):2427-2430
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裂解法合成2-(4-溴甲基苯基)丙酸 总被引:1,自引:0,他引:1
以2-(4-甲氧甲基苯基)丙酸甲酯为原料,利用氢溴酸裂解醚键高选择性制备了2-(4-溴甲基苯基)丙酸,避免了苄位甲基直接溴化产生多溴物、苯环溴化等副产物,收率达到67.1%,纯度99.2%。产品通过1H NMR的表征。同时,考察了物料配比、回流温度、反应时间对反应收率的影响,确定2-(4-甲氧基苯基)丙酸甲酯、47%氢溴酸和三溴化磷的摩尔比为1∶3∶1.5,回流温度55℃,反应9.5 h为最适宜工艺条件,简化了操作设备和操作过程,具有较高的工业化生产价值。 相似文献
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Karla Johanna Ruth Hoyer-Allo Martin Richard Spth Ruth Hanssen Marc Johnsen Susanne Brodesser Kathrin Kaufmann Katharina Kiefer Felix Carlo Koehler Heike Gbel Torsten Kubacki Franziska Grundmann Bernhard Schermer Jens Brüning Thomas Benzing Volker Burst Roman-Ulrich Müller 《International journal of molecular sciences》2021,22(11)
Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents, AKI can be effectively prevented through caloric restriction (CR), which has also been shown to increase lifespan in many species. In Caenorhabditis elegans (C. elegans), longevity studies revealed that a marked CR-induced reduction of endocannabinoids may be a key mechanism. Thus, we hypothesized that regulation of endocannabinoids, particularly arachidonoyl ethanolamide (AEA), might also play a role in CR-mediated protection from renal ischemia-reperfusion injury (IRI) in mammals including humans. In male C57Bl6J mice, CR significantly reduced renal IRI and led to a significant decrease of AEA. Supplementation of AEA to near-normal serum concentrations by repetitive intraperitoneal administration in CR mice, however, did not abrogate the protective effect of CR. We also analyzed serum samples taken before and after CR from patients of three different pilot trials of dietary interventions. In contrast to mice and C. elegans, we detected an increase of AEA. We conclude that endocannabinoid levels in mice are modulated by CR, but CR-mediated renal protection does not depend on this effect. Moreover, our results indicate that modulation of endocannabinoids by CR in humans may differ fundamentally from the effects in animal models. 相似文献
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Dr. Adam Daruzzaman Dr. Ian J. Clifton Dr. Robert M. Adlington Prof. Sir Jack E. Baldwin Dr. Peter J. Rutledge 《Chembiochem : a European journal of chemical biology》2013,14(5):599-606
Isopenicillin N synthase (IPNS) converts the linear tripeptide δ‐(L ‐α‐aminoadipoyl)‐L ‐cysteinyl‐D ‐valine (ACV) into bicyclic isopenicillin N (IPN) in the central step in the biosynthesis of penicillin and cephalosporin antibiotics. Solution‐phase incubation experiments have shown that IPNS turns over analogues with a diverse range of side chains in the third (valinyl) position of the substrate, but copes less well with changes in the second (cysteinyl) residue. IPNS thus converts the homologated tripeptides δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐valine (AhCV) and δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐allylglycine (AhCaG) into monocyclic hydroxy‐lactam products; this suggests that the additional methylene unit in these substrates induces conformational changes that preclude second ring closure after initial lactam formation. To investigate this and solution‐phase results with other tripeptides δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐Xaa, we have crystallised AhCV and δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐S‐methylcysteine (AhCmC) with IPNS and solved crystal structures for the resulting complexes. The IPNS:FeII:AhCV complex shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the IPNS:FeII:AhCmC complex, by virtue of thioether coordination to iron. AhCmC occupies two distinct conformations, both distorted relative to the natural substrate ACV, in order to accommodate the extra methylene group in the second residue. Attempts to turn these substrates over within crystalline IPNS using hyperbaric oxygenation give rise to product mixtures. 相似文献