PubMed Central (PMC3 - NLM DTD)
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Archive of life sciences journal literature at the U.S. National Institutes of Health (NIH), developed and managed by NIH's National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM).
Mostrando recursos 41 - 60 de 59,293
41.
Function of the C-terminus of ?29 DNA polymerase in DNA and terminal protein binding - Truniger, Verónica; Lázaro, José M.; Salas, Margarita
The thumb subdomain, located in various family B DNA polymerases in the C-terminal region, has been shown in their crystal structures to move upon binding of DNA, changing its conformation to nearly completely wrap around the DNA. It has therefore been involved in DNA binding. In agreement with this, partial proteolysis studies of ?29 DNA polymerase have shown that the accessibility of the cleavage sites located in their C-terminal region is reduced in the presence of DNA or terminal protein (TP), indicating that a conformational change occurs in this region upon substrate binding and suggesting that this region might be...
43.
Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: a comparison of tricyclo-DNA with LNA - Ittig, Damian; Liu, Songkai; Renneberg, Dorte; Schümperli, Daniel; Leumann, Christian J.
The nuclear antisense properties of a series of tricyclo (tc)-DNA oligonucleotide 915mers, targeted against the 3? and 5? splice sites of exon 4 of cyclophilin A (CyPA) pre-mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA-oligonucleotides. While the 9mers showed no significant antisense effect, the 1115mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence- and dose-dependent manner, as revealed by a RTPCR assay. The antisense efficacy of the tc-oligonucleotides was found to be superior to that of the LNA-oligonucleotides in all cases by...
44.
Triplex-forming oligonucleotide target sequences in the human genome - Goñi, J. Ramon; de la Cruz, Xavier; Orozco, Modesto
The existence of sequences in the human genome which can be a target for triplex formation, and accordingly are candidates for anti-gene therapies, has been studied by using bioinformatics tools. It was found that the population of triplex-forming oligonucleotide target sequences (TTS) is much more abundant than that expected from simple random models. The population of TTS is large in all the genome, without major differences between chromosomes. A wide analysis along annotated regions of the genome allows us to demonstrate that the largest relative concentration of TTS is found in regulatory regions, especially in promoter zones, which suggests a...
45.
Stereoselective excision of thymine glycol from oxidatively damaged DNA - Miller, Holly; Fernandes, Andrea S.; Zaika, Elena; McTigue, Monica M.; Torres, M. Cecilia; Wente, Maryann; Iden, Charles R.; Grollman, Arthur P.
DNA damage created by reactive oxygen species includes the prototypic oxidized pyrimidine, thymine glycol (Tg), which exists in oxidatively damaged DNA as two diastereoisomeric pairs. In Escherichia coli, Saccharomyces cerevesiae and mice, Tg is preferentially excised by endonuclease III (Endo III) and endonuclease VIII (Endo VIII), yNTG1 and yNTG2, and mNTH and mNEIL1, respectively. We have explored the ability of these DNA glycosylases to discriminate between Tg stereoisomers. Oligonucleotides containing a single, chromatographically pure (5S,6R) or (5R,6S) stereoisomer of Tg were prepared by oxidation with osmium tetroxide. Steady-state kinetic analyses of the excision process revealed that Endo III, Endo VIII,...
46.
Nucleotide incorporation by human DNA polymerase ? opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine - Graziewicz, Maria A.; Sayer, Jane M.; Jerina, Donald M.; Copeland, William C.
Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol ?) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DEdG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DEdA adducts derived from both BaP...
47.
A novel assay to determine the sequence preference and affinity of DNA minor groove binding compounds - Thomas, Rita; Gonzalez, Carolyn; Roberts, Christopher; Botyanszki, Janos; Lou, Lillian; Michelotti, Emil F.
Sequence-specific binding in the minor groove of DNA by small molecules is a growing area of research with possible therapeutic relevance. By selectively binding to DNA sequences required by critical transcription factors, these small molecules could potentially modulate the expression levels of disease-causing genes. Precise targeting of a critical transcription factor of a selected gene requires an understanding of the preferred sequence of the DNA binding compound. As new compounds are being synthesized, there is a need to evaluate their DNA recognition profile. We sought to establish a procedure to determine sequence preference of compounds with previously unknown binding properties....
48.
Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays - Fixe, F.; Dufva, M.; Telleman, P.; Christensen, C. B. V.
A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray slides. Immobilized and hybridized...
49.
Improved full-length cDNA production based on RNA tagging by T4 DNA ligase - Clepet, Christian; Le Clainche, Isabelle; Caboche, Michel
Second-strand cDNA priming is a central problem for full-length characterization of transcripts. A new strategy using bacteriophage T4 DNA ligase and partially degenerate adapters is proposed for grafting a sequence tag to the end of polyribonucleotides. Based on this RNA tagging system and previously described protocols, a new method for full-length cDNA production has been implemented. Validation of the method is shown in Arabidopsis thaliana by the construction of a full-length cDNA library and the analysis of 154 clones and by 5?-RACEPCR run on a documented experimental system.
50.
Dual-regulated expression of C/EBP-? and BMP-2 enables differential differentiation of C2C12 cells into adipocytes and osteoblasts - Fux, Cornelia; Mitta, Barbara; Kramer, Beat P.; Fussenegger, Martin
CCAAT/enhancer-binding proteins (C/EBPs) as well as bone morphogenic proteins (BMPs) play essential roles in mammalian cell differentiation in shaping adipogenic and osteoblastic lineages in particular. Recent evidence suggested that adipocytes and osteoblasts share a common mesenchymal precursor cell phenotype. Yet, the molecular details underlying the decision of adipocyte versus osteoblast differentiation as well as the involvement of C/EBPs and BMPs remains elusive. We have engineered C2C12 cells for dual-regulated expression of human C/EBP-? and BMP-2 to enable independent transcription control of both differentiation factors using clinically licensed antibiotics of the streptogramin (pristinamycin) and tetracycline (tetracycline) classes. Differential as well as...
51.
Biological detection of low radiation doses by combining results of two microarray analysis methods - Mercier, G.; Berthault, N.; Mary, J.; Peyre, J.; Antoniadis, A.; Comet, J.-P.; Cornuejols, A.; Froidevaux, C.; Dutreix, M.
The accurate determination of the biological effects of low doses of pollutants is a major public health challenge. DNA microarrays are a powerful tool for investigating small intracellular changes. However, the inherent low reliability of this technique, the small number of replicates and the lack of suitable statistical methods for the analysis of such a large number of attributes (genes) impair accurate data interpretation. To overcome this problem, we combined results of two independent analysis methods (ANOVA and RELIEF). We applied this analysis protocol to compare gene expression patterns in Saccharomyces cerevisiae growing in the absence and continuous presence of...
52.
Specific cleavage of DNA molecules at RecA-mediated triple-strand structure - Shigemori, Yasushi; Oishi, Michio
A novel procedure to cleave DNA molecules at any desired base sequence is presented. This procedure is based upon our finding that double-stranded DNA molecules at a site where RecA-mediated triple-stranded DNA structure with a complimentary deoxyoligonucleotide is located can be cleaved by a single-strand specific nuclease, such as nuclease S1 or BAL31, between the first base at the 5? termini of the deoxyoligonucleotides and the nearest base proximal to the 5? termini. Accordingly, the sequence as well as the number of the cleavage sites to be cleaved can be custom designed by selecting deoxyoligonucleotides with specific base sequences for...
53.
Method to integrate multiple plasmids into the mycobacterial chromosome - Saviola, Beatrice; Bishai, William R.
In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene. The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site....
54.
SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development - Thiel, Thomas; Kota, Raja; Grosse, Ivo; Stein, Nils; Graner, Andreas
With the influx of various SNP genotyping assays in recent years, there has been a need for an assay that is robust, yet cost effective, and could be performed using standard gel-based procedures. In this context, CAPS markers have been shown to meet these criteria. However, converting SNPs to CAPS markers can be a difficult process if done manually. In order to address this problem, we describe a computer program, SNP2CAPS, that facilitates the computational conversion of SNP markers into CAPS markers. 413 multiple aligned sequences derived from barley ESTs were analysed for the presence of polymorphisms in 235 distinct...
55.
Superior 5? homogeneity of RNA from ATP-initiated transcription under the T7 ?2.5 promoter - Coleman, Tricia M.; Wang, Guocan; Huang, Faqing
Transcription from the commonly used GTP- initiating T7 class III promoter ?6.5 frequently produces heterogeneous RNA at both 3? and 5? ends. We demonstrate here that RNA transcripts from the T7 class II promoter ?2.5 have superior 5? homogeneity over those from the ?6.5 promoter, with comparable total RNA yields. The overall homogeneity of RNA transcripts is improved to different degrees depending on RNA sequences, although transcription under ?2.5 does not affect the 3? heterogeneity of RNA. In combination with 3? RNA trimming by DNAzymes or ribozymes, this ATP- initiated transcription system based on the T7 ?2.5 promoter can provide...
56.
A real-time PCR assay for DNA-methylation using methylation-specific blockers - Cottrell, Susan E.; Distler, Jürgen; Goodman, Nancy S.; Mooney, Suzanne H.; Kluth, Antje; Olek, Alexander; Schwope, Ina; Tetzner, Reimo; Ziebarth, Heike; Berlin, Kurt
DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit...
57.
Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses - Kohli, Manu; Rago, Carlo; Lengauer, Christoph; Kinzler, Kenneth W.; Vogelstein, Bert
Emerging evidence suggests that recombinant adeno-associated viral (rAAV) vectors can be used for specific gene targeting in human somatic cells. We have developed an rAAV vector construction procedure employing fusion PCR and a single cloning step that considerably simplifies the knockout process. We demonstrate its utility by disrupting genes at specific positions within human colon cancer cells as well as within immortalized normal epithelial cells. This technology should be broadly applicable to in vitro studies that require the manipulation of the human genome.
58.
Ligase-mediated construction of branched DNA strands: a novel DNA joining activity catalyzed by T4 DNA ligase - Mendel-Hartvig, Maritha; Kumar, Anil; Landegren, Ulf
Branched nucleic acid strands exist as intermediates in certain biological reactions, and bifurcating DNA also presents interesting opportunities in biotechnological applications. We describe here how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5? end but two distinct 3? ends that extend from the 2? and 3? carbons, respectively, of an internal nucleotide. The nature of the reaction products is investigated, and optimal reaction conditions are reported for the construction of branched oligonucleotides. We discuss the utility of these branched DNA nanostructures for gene detection.
59.
PCR-based positive hybridization to detect genomic diversity associated with bacterial secondary metabolism - Pomati, Francesco; Neilan, Brett A.
A PCR-based positive hybridization (PPH) method was developed to explore toxic-specific genes in common between toxigenic strains of Anabaena circinalis, a cyanobacterium able to produce saxitoxin (STX). The PPH technique is based on the same principles of suppression subtractive hybridization (SSH), although with the former no driver DNA is required and two tester genomic DNAs are hybridized at high stringency. The aim was to obtain genes associated with cyanobacterial STX production. The genetic diversity within phylogenetically similar strains of A.circinalis was investigated by comparing the results of the standard SSH protocol to the PPH approach by DNA-microarray analysis. SSH allowed...
60.
Revised UV extinction coefficients for nucleoside-5?-monophosphates and unpaired DNA and RNA - Cavaluzzi, Michael J.; Borer, Philip N.
Ultraviolet absorption provides the nearly universal basis for determining concentrations of nucleic acids. Values for the UV extinction coefficients of DNA and RNA rely on the mononucleotide values determined 3050 years ago. We show that nearly all of the previously published extinction coefficients for the nucleoside-5?-monophosphates are too large, and in error by as much as 7%. Concentrations based on complete hydrolysis and the older set of values are too low by ?4% for typical RNA and 23% for typical DNA samples. We also analyzed data in the literature for the extinction coefficients of unpaired DNA oligomers. Robust prediction of...
41.
