PubMed Central (PMC3 - NLM DTD)
(2,081,148 recursos)
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 161 - 180 de 59,293
161.
Relevance of UP elements for three strong Bacillus subtilis phage ?29 promoters - Meijer, Wilfried J. J.; Salas, Margarita
Various Escherichia coli promoters contain, in addition to the classical 35 and 10 hexamers, a third recognition element, named the UP element. Located upstream of the 35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase ? subunit (?CTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early ?A-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic...
162.
Determination of detection and quantification limits for SNP allele frequency estimation in DNA pools using real time PCR - Schwarz, Gerhard; Bäumler, Stefan; Block, Annette; Felsenstein, Friedrich G.; Wenzel, Gerhard
The quantification of single nucleotide polymorphism (SNP) allele frequencies in pooled DNA samples using real time PCR is a promising approach for large-scale diagnostics and genotyping. The limits of detection (LOD) and limits of quantification (LOQ) for mutant SNP alleles are of particular importance for determination of the working range, which, in the case of allele-specific real time PCR, can be limited by the variance of calibration data from serially diluted mutant allele samples as well as by the variance of the 100% wild-type allele samples (blank values). In this study, 3? and 10? criteria were applied for the calculation...
163.
Unlocking hidden genomic sequence - Keith, Jonathan M.; Cochran, Duncan A. E.; Lala, Gita H.; Adams, Peter; Bryant, Darryn; Mitchelson, Keith R.
Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and...
164.
Nuclear localization signal and cell synchrony enhance gene targeting efficiency in primary fetal fibroblasts - Mir, Bashir; Piedrahita, Jorge A.
The use of primary somatic cells in nuclear transfer procedure has opened a new opportunity to manipulate domestic animal genomes via homologous recombination. To date, while a few loci have been targeted in somatic cells using similar enrichment strategies as those used in mouse ES cells, there have been problems of low efficiency, mixed targeted and non-targeted cells within a colony and difficulties in cloning the cell after targeting. Utilizing the hypoxanthine guanine phosphoribosyl transferase (HPRT) as a test locus, it was determined that while no targeted colonies were identified using a conventional targeting construct, an average of 1 per...
165.
Semiconductor nanocrystal probes for human metaphase chromosomes - Xiao, Yan; Barker, Peter E.
To improve signal stability and quantitation, an optically stable, novel class of fluorophore for hybridization analysis of human metaphase chromosomes is demonstrated. Detection of hybridization sites in situ was based on fluorescence from streptavidin-linked inorganic crystals of cadmium selenide [(CdSe)ZnS]. Fluorescence of nanocrystal fluorophores was significantly brighter and more photostable than organic fluorophores Texas Red and fluorescein. Thus, semiconductor nanocrystal fluorophores offer a more stable and quantitative mode of fluorescence in situ hybridization (FISH) for research and clinical applications.
166.
Genomic shotgun array: a procedure linking large-scale DNA sequencing with regional transcript mapping - Li, Ling-Hui; Li, Jian-Chiuan; Lin, Yung-Feng; Lin, Chung-Yen; Chen, Chung-Yung; Tsai, Shih-Feng
To facilitate transcript mapping and to investigate alterations in genomic structure and gene expression in a defined genomic target, we developed a novel microarray-based method to detect transcriptional activity of the human chromosome 4q22-24 region. Loss of heterozygosity of human 4q22-24 is frequently observed in hepatocellular carcinoma (HCC). One hundred and eighteen well-characterized genes have been identified from this region. We took previously sequenced shotgun subclones as templates to amplify overlapping sequences for the genomic segment and constructed a chromosome-region-specific microarray. Using genomic DNA fragments as probes, we detected transcriptional activity from within this region among five different tissues. The...
167.
Reliability of gene expression ratios for cDNA microarrays in multiconditional experiments with a reference design - König, Rainer; Baldessari, Danila; Pollet, Nicolas; Niehrs, Christof; Eils, Roland
In a typical gene expression profiling experiment with multiple conditions, a common reference sample is used for co-hybridization with the samples to yield expression ratios. Differential expression for any other sample pair can then be calculated by assembling the ratios from their hybridizations with the reference. In this study we test the validity of this approach. Differential expression of a sample pair (i, j) was obtained in two ways: directly, by hybridizations of sample i versus j, and indirectly, by multiplying the expression ratios for hybridizations of sample i versus pool and pool versus sample j. We performed gene expression...
168.
Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution - Wong, Tuck Seng; Tee, Kang Lan; Hauer, Berhard; Schwaneberg, Ulrich
Sequence saturation mutagenesis (SeSaM) is a conceptually novel and practically simple method that truly randomizes a target sequence at every single nucleotide position. A SeSaM experiment can be accomplished within 23 days and comprises four steps: generating a pool of DNA fragments with random length, tailing the DNA fragments with universal base using terminal transferase at 3?-termini, elongating DNA fragments in a PCR to the full-length genes using a single-stranded template and replacing the universal bases by standard nucleotides. Random mutations are created at universal sites due to the promiscuous base-pairing property of universal bases. Using enhanced green fluorescence protein...
169.
Surfection: a new platform for transfected cell arrays - Chang, Fu-Hsiung; Lee, Chien-Hsin; Chen, Ming-Ta; Kuo, Chun-Chen; Chiang, Yi-Lin; Hang, Chi-Ying; Roffler, Steve
Efficient high-throughput expression of genes in mammalian cells can facilitate large-scale functional genomic studies. Towards this aim, we developed a simple yet powerful method to deliver genes into cells by cationic polymers on the surface of substrates. Transfection can be achieved by directly contacting nucleic acidcell mixtures with the cationic substrates, e.g. polyethylenimine/collagen-coated wells. This single-step matrix-surface- mediated transfection method, termed surfection, can efficiently deliver multiple plasmids into cells and can successfully assay siRNA-mediated gene silencing. This technology represents the easiest method to transfer combinations of genes in large-scale arrays, and is a versatile tool for live-cell imaging and cell-based...
170.
Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display - Swers, Jeffrey S.; Kellogg, Brenda A.; Wittrup, K. Dane
Homologous recombination in yeast can be exploited to reliably generate libraries of >107 transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5? homology to the cut vector and the second scFv PCR product lacked 3? homology to the cut vector, or both PCR products had both 5? and 3?...
171.
Simple cDNA normalization using kamchatka crab duplex-specific nuclease - Zhulidov, Pavel A.; Bogdanova, Ekaterina A.; Shcheglov, Alex S.; Vagner, Laura L.; Khaspekov, George L.; Kozhemyako, Valery B.; Matz, Mikhail V.; Meleshkevitch, Ella; Moroz, Leonid L.; Lukyanov, Sergey A.; Shagin, Dmitry A.
We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturationreassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNARNA hybrid duplexes compared with ss DNA and...
172.
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements - Yang, Allen S.; Estécio, Marcos R. H.; Doshi, Ketan; Kondo, Yutaka; Tajara, Eloiza H.; Issa, Jean-Pierre J.
We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15?000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but...
173.
Doxycyline regulation in a single retroviral vector by an autoregulatory loop facilitates controlled gene expression in liver cells - Kühnel, Florian; Fritsch, Corinna; Krause, Sabine; Mundt, Bettina; Wirth, Thomas; Paul, Yasmin; Malek, Nisar Peter; Zender, Lars; Manns, Michael Peter; Kubicka, Stefan
The tetracycline system has limitations in liver cells, such as toxic effects and low controllability. We generated different retroviral vectors for controlled gene expression in liver cells, in which the regulatory elements were arranged in different patterns. Only the organization of the tetracycline system in an autoregulatory loop in the sense orientation results in high retroviral titres and in tight regulation of gene expression in highly differentiated hepatoma cells. Because of the toxicity of the transactivator tTA, it was impossible to establish doxycycline-dependent stable HepG2 cell lines. To avoid sequelching-related toxicity in liver cells, we replaced tTA with new non-toxic...
174.
Large-scale determination of the methylation status of retrotransposons in different tissues using a methylation tags approach - Khodosevich, Konstantin; Lebedev, Yuri; Sverdlov, Eugene D.
A technique for simultaneous determination of the methylation status of numerous loci containing retroelements (REs) is reported. It is based on the observation that methylated and unmethylated areas in the genome are usually extended, and therefore the methylation of particular methyl-sensitive restriction endonuclease recognition sites might reflect the methylation status of DNA regions around them. The method includes dot-blot hybridization of repeat flanking sequences arrayed on a solid support with specifically amplified flanking regions of presumably unmethylated repeats. A multitude of flanking regions of REs adjacent to unmethylated restriction sites are amplified simultaneously, providing a complex hybridization probe. The technique...
175.
Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays - Di Giusto, Daniel A.; King, Garry C.
The effect of locked nucleic acid (LNA) modification position upon representative DNA polymerase and exonuclease activities has been examined for potential use in primer extension genotyping applications. For the 3??5? exonuclease activities of four proofreading DNA polymerases (Vent, Pfu, Klenow fragment and T7 DNA polymerase) as well as exonuclease III, an LNA at the terminal (L-1) position of a primer is found to provide partial protection against the exonucleases of the two family B polymerases only. In contrast, an LNA residue at the penultimate (L-2) position generates essentially complete nuclease resistance. The polymerase active sites of these enzymes also display...
176.
Generating in vitro transcripts with homogenous 3? ends using trans-acting antigenomic delta ribozyme - Wich?acz, Agnieszka; ??giewicz, Micha?; Ciesio?ka, Jerzy
In most in vitro run-off transcription reactions with T7 RNA polymerase, transcripts with heterogeneous ends are commonly obtained. Towards the goal of finding a simple and effective procedure for correct processing of their 3? ends we propose the use of trans-acting antigenomic delta ribozyme. We demonstrate that the extension of nascent transcripts with only seven nucleotides complementary to the ribozymes recognition site, and subsequently, the removal of those nucleotides with the ribozyme acting in trans, is an efficient procedure for generating transcripts with homogenous 3? ends. This approach was tested on two model RNA molecules: an in vitro transcript of...
177.
LSimpute: accurate estimation of missing values in microarray data with least squares methods - Bø, Trond Hellem; Dysvik, Bjarte; Jonassen, Inge
Microarray experiments generate data sets with information on the expression levels of thousands of genes in a set of biological samples. Unfortun ately, such experiments often produce multiple missing expression values, normally due to various experimental problems. As many algorithms for gene expression analysis require a complete data matrix as input, the missing values have to be estimated in order to analyze the available data. Alternatively, genes and arrays can be removed until no missing values remain. However, for genes or arrays with only a small number of missing values, it is desirable to impute those values. For the subsequent...
178.
Direct interaction of NRSF with TBP: chromatin reorganization and core promoter repression for neuron-specific gene transcription - Murai, Kiyohito; Naruse, Yoshihisa; Shaul, Yosef; Agata, Yasutoshi; Mori, Nozomu
Neural restrictive silencer factor, NRSF (also known as REST) binds a neuronal cell type selective silencer element to mediate transcriptional repression of neuron-specific genes in non-neuronal cells and neuronal progenitors. Two repression domains (RD-1 and RD-2) occur in its N-terminal and C-terminal regions, respectively. RD-1 recruits mSin3 and HDAC, thereby inhibiting transcription by inducing reorganization of the chromatin structure. However, little is known about how such global repression becomes promoter-specific repression or whether the NRSFHDAC complex can interact with transcriptional core factors at each specific promoter. Here we show evidence that NRSF interacts with core promoter factors, including TATA-binding protein...
179.
Genome-wide identification of genes likely to be involved in human genetic disease - López-Bigas, Núria; Ouzounis, Christos A.
Sequence analysis of the group of proteins known to be associated with hereditary diseases allows the detection of key distinctive features shared within this group. The disease proteins are characterized by greater length of their amino acid sequence, a broader phylogenetic extent, and specific conservation and paralogy profiles compared with all human proteins. This unique property pattern provides insights into the global nature of hereditary diseases and moreover can be used to predict novel disease genes. We have developed a computational method that allows the detection of genes likely to be involved in hereditary disease in the human genome. The...
180.
PeerGAD: a peer-review-based and community-centric web application for viewing and annotating prokaryotic genome sequences - DAscenzo, Mark D.; Collmer, Alan; Martin, Gregory B.
PeerGAD is a web-based database-driven application that allows community-wide peer-reviewed annotation of prokaryotic genome sequences. The application was developed to support the annotation of the Pseudomonas syringae pv. tomato strain DC3000 genome sequence and is easily portable to other genome sequence annotation projects. PeerGAD incorporates several innovative design and operation features and accepts annotations pertaining to gene naming, role classification, gene translation and annotation derivation. The annotator tool in PeerGAD is built around a genome browser that offers users the ability to search and navigate the genome sequence. Because the application encourages annotation of the genome sequence directly by researchers...
161.
