Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.
- MeSH
- 5-methylcytosin * MeSH
- deoxyribonukleosidy MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- epigeneze genetická MeSH
- nukleotidy metabolismus MeSH
- pyrimidinové nukleotidy * MeSH
- pyrimidiny MeSH
- restrikční enzymy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Five 2-substituted 2'-deoxyinosine triphosphates (dRITP) were synthesized and tested as substrates in enzymatic synthesis of minor-groove base-modified DNA. Only 2-methyl and 2-vinyl derivatives proved to be good substrates for Therminator DNA polymerase, whilst all other dRITPs and other tested DNA polymerases did not give full length products in primer extension. The DNA containing 2-vinylhypoxanthine was then further modified through thiol-ene reactions with thiols. Cross-linking reaction between cysteine-containing minor-groove binding dodecapeptide and DNA proceeded thanks to the proximity effect between thiol and vinyl groups inside the minor groove. 2-Substituted dIRTPs and also previously prepared 2-substituted 2'-deoxyadenosine triphosphates (dRATP) were then used for enzymatic synthesis of minor-groove modified DNA to study the effect of minor-groove modifications on cleavage of DNA by type II restriction endonucleases (REs). Although the REs should recognize the sequence through H-bonds in the major groove, some minor-groove modifications also had an inhibiting effect on the cleavage.
- MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA biosyntéza chemie MeSH
- inosintrifosfát analogy a deriváty chemická syntéza metabolismus MeSH
- konformace nukleové kyseliny MeSH
- restrikční endonukleasy typu II metabolismus MeSH
- restrikční enzymy metabolismus MeSH
- substrátová specifita * MeSH
- vinylové sloučeniny chemie MeSH
- vodíková vazba MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Here, we present an improved amplified fragment length polymorphism (AFLP) protocol using restriction enzymes (AscI and SbfI) that recognize 8-base pair sequences to provide alternative optimization suitable for species with a genome size over 70 Gb. This cost-effective optimization massively reduces the number of amplified fragments using only +3 selective bases per primer during selective amplification. We demonstrate the effects of the number of fragments and genome size on the appearance of nonidentical comigrating fragments (size homoplasy), which has a negative impact on the informative value of AFLP genotypes. We also present various reaction conditions and their effects on reproducibility and the band intensity of the extremely large genome of Viscum album. The reproducibility of this octo-cutter protocol was calculated using several species with genome sizes ranging from 1 Gb (Carex panicea) to 76 Gb (V. album). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes (V. album, Galanthus nivalis and Pinus pumila).
- MeSH
- analýza polymorfismu délky amplifikovaných restrikčních fragmentů metody MeSH
- DNA rostlinná genetika metabolismus MeSH
- genom rostlinný * MeSH
- genotypizační techniky metody MeSH
- reprodukovatelnost výsledků MeSH
- restrikční enzymy metabolismus MeSH
- rostliny klasifikace genetika MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
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.
- MeSH
- DNA biosyntéza chemie metabolismus MeSH
- restrikční enzymy metabolismus MeSH
- štěpení DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We present a DNA-based implementation of reaction system with molecules encoding elements of the propositional logic, that is, propositions and formulas. The protocol can perform inference steps using, for example, modus ponens and modus tollens rules and de Morgan's laws. The set of the implemented operations allows for inference of formulas using the laws of natural deduction. The system can also detect whether a certain proposition a can be deduced from the basic facts and given rules. The whole protocol is fully autonomous; that is, after introducing the initial set of molecules, no human assistance is needed. Only one restriction enzyme is used throughout the inference process. Unlike some other similar implementations, our improved design allows representing simultaneously a fact a and its negation ~a, including special reactions to detect the inconsistency, that is, a simultaneous occurrence of a fact and its negation. An analysis of correctness, completeness, and complexity is included.
Green tea polyphenols (GTP) are widely believed to function as antioxidants and antimicrobial agents. Here we observed that GTP and epigallocatechin gallate, the most abundant catechin in GTP, could also function as prooxidants and produce hydrogen peroxide (H2O2) to inhibit the growth of Pseudomonas aeruginosa. pH value of the medium was the key factor that affected prooxidant versus antioxidant property of GTP. Under weakly acidic conditions (pH 5.5-6.5), GTP showed antioxidant activity by eliminating H2O2; whereas, under neutral and weakly alkaline conditions (pH 7.0-8.0), GTP showed prooxidant activity and inhibited the growth of P. aeruginosa. Furthermore, we studied the effects of GTP on gene expression profiles of a few oxidative stress-related genes by quantitative real-time PCR analysis. After 10 min to 1 h of exposure under weakly alkaline condition, GTP significantly up-regulated expression levels of katB, sodM, ohr, lexA, and recN gene. These findings highlight that the pH-dependent H2O2 production by GTP contributes to the antibacterial activity and can induce oxidative stress-related responses in P. aeruginosa.
- MeSH
- antibakteriální látky farmakologie MeSH
- antioxidancia farmakologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- čaj chemie MeSH
- katechin analogy a deriváty farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- oxidační stres účinky léků MeSH
- peroxid vodíku analýza metabolismus MeSH
- polyfenoly analýza farmakologie MeSH
- Pseudomonas aeruginosa účinky léků růst a vývoj MeSH
- reaktivní formy kyslíku analýza farmakologie MeSH
- restrikční enzymy genetika metabolismus MeSH
- rostlinné extrakty analýza farmakologie MeSH
- serinové endopeptidasy genetika metabolismus MeSH
- upregulace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Two hundred eighty-four isolates of enterococci from feces of wild living chamois from alpine environments were tested for sensitivity to three antibiotics. Low frequency of resistance was observed in studied enterococcal populations (about 5 % for tetracycline and erythromycin and 0 % for ampicillin). In six animals, the population of enterococci lacked any detectable resistance. Our data indicated that enterococcal population in feces of the majority of studied animals did not encounter mobile genetic elements encoding antibiotic resistance probably due to spatial separation and/or due to low exposure to the antibiotics. Based on resistance profiles observed, three populations were analyzed for the presence of restriction endonucleases. The restriction enzymes from two isolates-31K and 1K-were further purified and characterized. Restriction endonuclease Efa1KI recognizes CCWGG sequence and is an isoschizomer of BstNI. Endonuclease Efc31KI, a BsmAI isoschizomer, recognizes the sequence GTCTC and it is a first restriction endonuclease identified in Enterococcus faecium. Our data indicate that restriction-modification (R-M) systems do not represent an efficient barrier for antibiotic resistance spreading; enterococcal populations colonized by antibiotics resistance genes were also colonized by the R-M systems.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- Enterococcus účinky léků enzymologie genetika izolace a purifikace MeSH
- feces mikrobiologie MeSH
- mikrobiální testy citlivosti MeSH
- restrikční enzymy chemie genetika metabolismus MeSH
- Rupicapra mikrobiologie MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.
- MeSH
- Cicer mikrobiologie MeSH
- DNA fingerprinting metody MeSH
- DNA fungální genetika chemie MeSH
- DNA primery genetika MeSH
- financování organizované MeSH
- Fusarium genetika izolace a purifikace klasifikace MeSH
- genetická variace MeSH
- mezerníky ribozomální DNA genetika chemie MeSH
- molekulární sekvence - údaje MeSH
- mykologie metody MeSH
- nemoci rostlin mikrobiologie MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- restrikční enzymy metabolismus MeSH
- sekvenční analýza DNA MeSH
- senzitivita a specificita MeSH
- Geografické názvy
- Indie MeSH
Components of plant essential oils have been reported to have health benefit properties, including antioxidative, anti-tumour, antimicrobial, anti-stress, and immunomodulative activities. We examined the anti-inflammatory effects of thymoquinone, the active ingredient in the volatile oil of Nigella sativa seeds, and borneol, the active component of Salvia officinalis essential oil, on TNBS-induced colitis in mice. Thymoquinone was added to the commercial diet at a concentration of 0.05 % and borneol at two concentrations (0.09% and 0.18%) and fed to ICR mice 5 days before induction of TNBS colitis. Seven days after TNBS administration the mice were killed and macroscopic and histological scores were evaluated. Cytokine mRNA expression in colonic tissue was assessed using quantitative realtime RT-PCR. We did not detect any significant changes in macroscopic and histological scores between experimental and control groups, but we observed a significant decrease in proinflammatory cytokine (IL-1beta and IL-6) mRNA expression in colon tissue in the 0.09% and 0.18% borneol-treated groups of mice in comparison to the control group. Surprisingly, we were not able to confirm anti-inflammatory effects of thymoquinone in TNBS colitis. In conclusion, our data show that borneol is able to significantly suppress proinflammatory cytokine mRNA expression in colonic inflammation, although no significant morphological changes are visible.
- MeSH
- benzochinony farmakologie MeSH
- bornany farmakologie MeSH
- cytokiny genetika metabolismus MeSH
- DNA metabolismus MeSH
- kolitida chemicky indukované patologie MeSH
- kolon patologie účinky léků MeSH
- kyselina trinitrobenzensulfonová farmakologie MeSH
- messenger RNA genetika metabolismus MeSH
- myši MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese účinky záření MeSH
- restrikční enzymy metabolismus MeSH
- tělesná hmotnost účinky léků MeSH
- velikost orgánu účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
