Thymine Research Articles

Impact of DNA–Surface Interactions on the Stability of DNA Hybrids

The structure and stability of single- and double-stranded DNA hybrids immobilized on gold are strongly affected by nucleotide-surface interactions. To systematically analyze the effects of these interactions, a set of model DNA hybrids was prepared in conformations that ranged from end-tethered double-stranded to directly adsorbed single-stranded (hairpins) and characterized by surface plasmon resonance (SPR) imaging, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. The stabilities of these hybrids were evaluated by exposure to a series of stringency rinses in solutions of successively lower ionic strength and by competitive hybridization experiments. In all cases, directly adsorbed DNA hybrids are found to be significantly less stable than either free or end-tethered hybrids. The surface-induced weakening and the associated asymmetry in hybridization responses of the two strands forming hairpin stems are most pronounced for single-stranded hairpins containing blocks of m adenine (A) nucleotides and n thymine (T) nucleotides, which have high and low affinity for gold surfaces, respectively. The results allow a qualitative scale of relative stabilities to be developed for DNA hybrids on surfaces. Additionally, the results suggest a route for selectively weakening portions of immobilized DNA hybrids and for introducing asymmetric hybridization responses by using sequence design to control nucleotide-surface interactions--a strategy that may be used in advanced biosensors and in switches or other active elements in DNA-based nanotechnology.

SiC nanoparticles-modified glassy carbon electrodes for simultaneous determination of purine and pyrimidine DNA bases

For the first time a novel and simple electrochemical method was used for simultaneous detection of DNA bases (guanine, adenine, thymine and cytosine) without any pretreatment or separation process. Glassy carbon electrode modified with silicon carbide nanoparticles (SiCNP/GC), have been used for electrocatalytic oxidation of purine (guanine and adenine) and pyrimidine bases (thymine and cytosine) nucleotides. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) techniques were used to examine the structure of the SiCNP/GC modified electrode. The modified electrode shows excellent electrocatalytic activity toward guanine, adenine, thymine and cytosine. Differential pulse voltammetry (DPV) was proposed for simultaneous determination of four DNA bases. The effects of different parameters such as the thickness of SiC layer, pulse amplitude, scan rate, supporting electrolyte composition and pH were optimized to obtain the best peak potential separation and higher sensitivity. Detection limit, sensitivity and linear concentration range of the modified electrode toward proposed analytes were calculated for, guanine, adenine, thymine and cytosine, respectively. As shown this sensor can be used for nanomolar or micromolar detection of different DNA bases simultaneously or individually. This sensor also exhibits good stability, reproducibility and long lifetime.

Disinfection of ocular cells and tissues by atmospheric-pressure cold plasma

Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. Our previous studies (1) demonstrated that atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas inactivated ocular pathogens in vitro. This study explored whether APCP inactivates ocular pathogens without causing significant tissue damage. We tested the APCP effects on cultured Pseudomonas aeruginosa, Staphylococcus aureus, A. fumigatus, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo cornea. Exposure to APCP for 0.5–5 min significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay and Tunel analysis). Since our previous study indicated that exposure to plasma increases intracellular reactive oxygen species (ROS) production, ROS levels in APCP exposed microorganisms and keratocytes were analyzed by 2’,7’-dichlorofluorescein diacetate (HDCF-DA) fluorescence. The potential genotoxic effects of plasma on cells and tissues were evaluated by analyses of thymine dimers (TD), genes and proteins involved in DNA damage and repair (OGG1, GPX, NRF2) at set time intervals. High levels of intracellular reactive oxygen species (ROS) were found in exposed microorganisms and cells. Immunoassay confirmed no induction of thymine dimers in corneal tissues. Conversely, a transient expression of genes and proteins recruited following oxidative stress was determined in ocular cells and corneas by qRT-PCR and Western blotting. In conclusion, a short application of APCP appears to be an efficient and rapid ocular disinfectant with ROS production likely causing pathogen killing and no substantial effects on ocular cells and tissues. The same APCP treatment to conjuntival fibroblasts and keratocytes caused a time-restricted formation of ROS and a change in some stress-response genes.

Characterization of two Egyptian native chicken breeds using genetic and immunological parameters

In order to identify and characterize our native chicken breeds we used two approaches, one of them is genetic and the other concerns with the immunological status of the chickens. In this study, the first 539 bases of the mtDNA D-loop region of two Egyptian native breeds (Fayoumi and Dandarawi, from El-Fayoum research station) were amplified and sequenced. The alignment results showed an approximate tandem repeat of 60-base units with the first 34 nucleotides being exact. These 34-base units were completely identical and conserved in both Egyptian and GenBank database samples. The multiple alignment results showed also that there are three transversions specific for the Egyptian breeds only. Two of them are not specific for certain breed since both of the two breeds showed the normal and mutated nucleotide. The third transversions seems to be specific to Egyptian Dandarawi breed only. The first mutation site is at the position (457) where Adenine nucleotide transversed to Cytosine nucleotide. The second mutation site is at the position (464) where Guanine nucleotide transversed to Thymine nucleotide. The third mutation site is at the position (483) where Adenine nucleotide transversed to Thymine nucleotide. With respect to immunological parameters, it could be speculated that the Fayoumi strain had hyper responders to phytohemagglutinin-P (PHA-P) injection compared to Dandarawi ones. Opposite trend was noticed for anti-SRBCs antibody response.The results may give an insight into the genetic differentiation and immunological status of the Egyptian domestic fowl. The results did not show direct relation between the two approaches used. However, the results of the two approaches can complete the identification and characterization of the chickens. Also, they could be used in future as bases for more studies.

Polymer supported calixarene derivative useful for solid-phase synthesis application

A calix[4]arene derivative has been anchored to carboxyl CPG and TentaGel supports by an easily cleavable ester bond and DMT groups allow a simple loading evaluation via UV–vis spectroscopy. The loading of the calixarene on TentaGel resin has also been estimated by HR-MAS NMR experiments. The potential of the polymer supported calixarenes ( 9 and 10) in solid phase synthesis has been tested by condensation of four thymine nucleotide units onto the upper rim of the calix[4]arene skeleton.

ChemInform Abstract: Synthesis of 3′-C-Ethynylnucleosides of Thymine.

ChemInform Abstract: Synthesis of 3′-C-Ethynylnucleosides of Thymine.

Incorporation of Thymine Nucleotides by DNA Polymerases through T–HgII–T Base Pairing

DNA in schlechter Gesellschaft: In Gegenwart von HgII-Ionen führten DNA-Polymerasen Thymidin-5′-triphosphat (TTP) gegenüber einem Thyminrest im Templatstrang ein bildeten zur Verlängerung des Primerstrangs eine Phosphodiesterbindung. DNA-Polymerasen erkannten dieses ungewöhnliche, durch ein Metallion verknüpfte Basenpaar und synthetisierten darüber hinweg das Volllängenprodukt (siehe Bild).

Identification of the TcpP-Binding Site in the toxT Promoter of Vibrio cholerae and the Role of ToxR in TcpP-Mediated Activation

ToxR-dependent recruitment of TcpP to the toxT promoter facilitates toxT transcription in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin expression and secretion. Although TcpP usually requires ToxR to activate the toxT promoter, TcpP overexpression can circumvent the requirement for ToxR in this process. To define nucleotides critical for TcpP-dependent promoter recognition and activation, a series of toxT promoter derivatives with single-base-pair transversions spanning the TcpP-binding site were generated and used as plasmid-borne toxT-lacZ fusions, as DNA mobility shift targets, and as allelic replacements of the chromosomal toxT promoter. When present in ΔtoxR V. cholerae overexpressing TcpP, several transversions affecting nucleotides within two direct repeats present in the TcpP-binding region (TGTAA-N(6)-TGTAA) caused defects in TcpP-dependent toxT-lacZ fusion activation and toxin production. Electrophoretic mobility shift assays demonstrated that these same transversions reduced the affinity of the toxT promoter for TcpP. The presence of ToxR suppressed transcription activation defects associated with most, but not all, transversions. Particularly, the central thymine nucleotide of both pentameric repeats was essential for efficient toxT activation, even in the presence of ToxR. These results suggest that the toxT promoter recognition function provided by ToxR can facilitate the interaction of TcpP with the toxT promoter but is insufficient for promoter activation when the TcpP-binding site has been severely compromised by mutation. Thus, the interaction of TcpP with nucleotides of the direct repeat sequences appears to be a prerequisite for toxT promoter activation.

Selective alkylation at the N1 position of thymine and uracil in the Mitsunobu system

Selective alkylation at the N1 position of thymine and uracil in the Mitsunobu system

Incorporation of Thymine Nucleotides by DNA Polymerases through T–HgII–T Base Pairing

Incorporation of Thymine Nucleotides by DNA Polymerases through T–Hg^II–T Base Pairing

Antitumor activity of bent metallocenes: electronic structure analysis using DFT computations

The antitumor activities of bent metallocenes [Cp-M-Cp](2+) (M = Ti, V, Nb, Mo) and complexes of them with guanine, adenine, thymine and cytosine nucleotides have been probed using electronic structure calculations. DFT/BP86 calculations have revealed that the bent metallocene-nucleotide interaction strongly depends on the stability of the hydrolyzed form of the bent metallocene dichloride [Cp(2)M](2+) species, and in turn the stability of the [Cp(2)M](2+) species strongly depends on the electronic structure of [Cp(2)M](2+). Detailed electronic structure and Walsh energy analyses have been carried out for the hydrolyzed forms of four [Cp-M-Cp](2+) (M = Ti, V, Nb, Mo) species to find out why the bent structure is unusually stable. Energy changes that occur during the bending process in frontier molecular orbitals as well as the p(π)-d(π) overlap have been invoked to account for the anticipated antitumor activities of these species. The bonding situation and the interactions in bent metallocene-nucleotide adducts were elucidated by fragment analysis. Of the four nucleotides complexed with the four bent metallocenes, adenine and guanine show better binding abilities than the other two nucleotides. Metallocenes of second-row transition metals exhibit better binding with pyrimidine-base nucleotides. In particular, the Lewis acidic bent metallocenes interact strongly with nucleotides. The antitumor activity is directly related to the binding strength of the bent metallocene with nucleotide adducts, and the computed interaction energy values correlate very well with the experimentally observed antitumor activities.

Reaction of peroxyl radicals with uracils in the event of inhibition of isopropyl alcohol oxidation

99 A number of pyrimidine derivatives (6 methyl uracil, 5 fluorouracil, 5 hydroxy 6 methyluracil) are pharmaceuticals exhibiting a wide spectrum of activ ity. The efficacy of pharmaceuticals as antioxidants is one of the main characteristics of their biological activity. However, the mechanism of inhibition of free radical induced chain oxidation by uracils has been poorly studied. The primary event of attack of poten tial antioxidant molecule by peroxyl radical plays an important role in the mechanism of oxidation retarda tion because it affects further reaction course. It is known [1] that the free radical oxidation of virtually equimolar amounts of 2,2' azobis(2 methylpropion amide) dihydrochloride and 5 methyluracil with atmospheric oxygen for 25 h at 313 K leads to the fol lowing products:

Meta‐analysis of cancer incidence in folic acid supplementation trials

It has been suggested by several research groups that folate is procarcinogenic. Data in the 1940s of folate supplementation indicated that it enhanced the proliferation of cancer cells. Folate is a unique vitamin because its coenzymes are required for de novo adenine, guanine, and thymine nucleotide biosynthesis; therefore, it is essential for DNA synthesis and repair, and RNA synthesis, and structurally related folate analogs, such as methotrexate, are used as anticancer agents. Using a meta‐analysis weighted for the duration of folic acid (FA, pteroylglutamic acid) supplementation, we analyzed the cancer incidence of 5 published large prospective FA supplementation trials that lasted 1 year or more involving men and women. These articles were selected from over 1,100 identified by a PubMed search that was done in June 2009, and skin cancer was excluded by the original investigators or by us. Our data suggest that cancer incidence was higher in the FA supplemented groups than the non‐FA supplemented groups (relative risk = 1.26 [95% confidence interval: 1.05–1.51]). FA supplementation trials should be performed with careful monitoring of cancer incidence by investigators, and funding agencies should provide long‐term support. In the US, solid monitoring systems to detect side effects, such as increased cancer risk, should have been established before the food FA fortification mandate.

Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures: IMPLICATIONS FOR GENOMIC INSTABILITY

The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C((d))C((S))C((S))" (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability.

Probing the structure of Leishmania donovani chagasi DHFR-TS: comparative protein modeling and protein–ligand interaction studies

Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. It also acts as a drug target for Leishmaniasis. Inhibition of DHFR leads to cell death through lack of thymine (nucleotide metabolism). Although the crystal structures of Leishmania major and Trypanosoma cruzi DHFR-thymidylate synthase (TS) have been resolved, to date there is no three-dimensional (3D)-structural information on DHFR-TS of Leishmania donovani chagasi, which causes visceral leishmaniasis. Our aim in this study was to model the 3D structure of L. donovani chagasi DHFR-TS, and to investigate the structural requirements for its inhibition. In this paper we describe a highly refined homology model of L. donovani chagasi DHFR-TS based on available crystallographic structures by using the Homology module of Insight II. Structural refinement and minimization of the generated L. donovani chagasi DHFR-TS model employed the Discover 3 module of Insight II and molecular dynamic simulations. The model was further validated through use of the PROCHECK, Verify_3D, PROSA, PSQS and ERRAT programs, which confirm that the model is reliable. Superimposition of the model structure with the templates L. major A chain, L. major B chain And T. cruzi A chain showed root mean square deviations of 0.69 A, 0.71 A and 1.11 A, respectively. Docking analysis of the L. donovani chagasi DHFR-TS model with methotrexate enabled us to identify specific residues, viz. Val156, Val30, Lys95, Lys75 and Arg97, within the L. donovani chagasi DHFR-TS binding pocket, that play an important role in ligand or substrate binding. Docking studies clearly indicated that these five residues are important determinants for binding as they have strong hydrogen bonding interactions with the ligand.

Sequence analysis of STK11 gene in a patient with Peutz-Jeghers syndrome.

Objective To analyze the STK11 gene mutation in a sporadic Chinese Datient with Peutz Jeghers syndrome(PJS)so as to provide a basis for the genetic diagnosis and counseling of PJS.Methods Whole blood samples were obtained from a female patient with PJS,her parents and sister.as well as from 100 unrelated,normal individuals as control.Genomic DNA was extracted,and the whole coding region of STK11 gene was amplified by PCR followed by direct sequencing.Results Molecular analysis revealed a novel het-erozygous mutation C73S in the patient,which resulted from the substitution of thymine(T)for adenine(A) at codon 217 in exon 1 of STK11 gene.However, the novel mutation was not found in unaffected family mem-bers or unrelated controls.Conclusion A novel missense mutation C73S,which may contribute to the devel-opment of PJS,is found in the patient. Key words: Peutz-Jeghers syndrome; Gene,STK11; Gene,LKB1; Mutation

Zinc-cyclen coordination to UTP, TTP or pyrophosphate induces pyrene excimer emission

Pyrene labelled Zn(2+)-cyclen 1 and bis-Zn(2+)-bis-cyclen 2 complexes were synthesized. The reversible coordination at physiological pH of Zn(2+)-cyclens to phosphate anions and to imide moieties, as present in thymine and uracil nucleotides, is well known. In the presence of analytes bearing a phosphate and an imide or two phosphate groups the formation of a ternary complex consisting of two pyrene-labelled metal complexes and the analyte molecule, is observed. The close proximity of the pyrene labels in the complex induces pyrene excimer emission, which is observable by the unarmed eye. By this, the presence of UMP, UDP, UTP and TTP in buffered aqueous solution is signalled, while other nucleotides are not able to induce excimer emission. In the same way, Zn(2+)-cyclen-pyrene acts as luminescent chemosensor for PP(i) and fructose-1,6-bisphosphate in aqueous buffer.

Thymine molecule excitation by slow electrons

В статті представлено результати по дослідженню збудження молекули тиміну повільними електронами. Електронний пучок з величиною струму 300 нА моноенергетичністю 150 меВ формувався гіпоциклоїдальним електронним монохроматором (ГЕМ). З використанням паронаповненої комірки виміряно спектр випромінювання в області 300-550 нм та оптичні функції збудження (ОФЗ) чотирьох найбільш інтенсивних смуг випромінювання.

A patient with Smith–Lemli–Opitz syndrome: novel mutation of the DHCR7 gene and effects of therapy with simvastatin and cholesterol supplement

The Smith-Lemli-Opitz (SLO) syndrome is a multiple congenital anomaly with mental retardation due to a decreased or lack of activity of 7-dehydrocholesterol reductase as a consequence of mutations of the DHCR7 gene. This paper describes a special patient with SLO syndrome. Laboratory examination showed low cholesterol (2.77 mmol/L) and increased 7-dehydrocholesterol level (102 mg/L). Molecular genetic analysis revealed a compound heterozygosity c.964-1G>C/p.G366V (c.G1370T) of the proband. The p.G366V is a novel mutation of the DHCR7 gene with guanine by thymine nucleotide exchange resulting in glycin by valin amino acid exchange in the dehydrocholesterol reductase enzyme. Simvastatin (0.2 mg/kg/day) and cholesterol replacement therapy (150-250 mg/kg/day) led to significant improvement in the patient's laboratory findings (7-dehydrocholesterol, cholesterol) as well as in his behavior and gross motor function. Our patient demonstrates that the c.964-1G>C/p.G366V (c.G1370T) genotype of combined heterozygosity is associated with a typical form of SLO syndrome along with moderately altered laboratory findings and a favorable biochemical response to cholesterol and simvastatin treatment.

Human Male Infertility Caused by Mutations in the CATSPER1 Channel Protein

Male infertility, a common barrier that prevents successful conception, is a reproductive difficulty affecting 15% of couples. Heritable forms of nonsyndromic male infertility can arise from single-gene defects as well as chromosomal abnormalities. Although no CATSPER gene has been identified as causative for human male infertility, male mice deficient for members of the CatSper gene family are infertile. In this study, we used routine semen analysis to identify two consanguineous Iranian families segregating autosomal-recessive male infertility. Autozygosity by descent was demonstrated in both families for a approximately 11 cM region on chromosome 11q13.1, flanked by markers D11S1765 and D11S4139. This region contains the human CATSPER1 gene. Denaturing high-performance liquid chromatography (DHPLC) and bidirectional sequence analysis of CATSPER1 in affected family members revealed two separate insertion mutations (c.539-540insT and c.948-949insATGGC) that are predicted to lead to frameshifts and premature stop codons (p.Lys180LysfsX8 and p.Asp317MetfsX18). CATSPER1 is one of four members of the sperm-specific CATSPER voltage-gated calcium channel family known to be essential for normal male fertility in mice. These results suggest that CATSPER1 is also essential for normal male fertility in humans.