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 61 - 80 de 59,293
61.
Formation of an intramolecular triple-stranded DNA structure monitored by fluorescence of 2-aminopurine or 6-methylisoxanthopterin - Shchyolkina, Anna K.; Kaluzhny, Dmitry N.; Borisova, Olga F.; Hawkins, Mary E.; Jernigan, Robert L.; Jovin, Thomas M.; Arndt-Jovin, Donna J.; Zhurkin, Victor B.
The parallel (recombination) R-triplex can accommodate any nucleotide sequence with the two identical DNA strands in parallel orientation. We have studied oligonucleotides able to fold back into such a recombination-like structure. We show that the fluorescent base analogs 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI) can be used as structural probes for monitoring the integrity of the triple-stranded conformation and for deriving the thermodynamic characteristics of these structures. A single adenine or guanine base in the third strand of the triplex-forming and the control oligonucleotides, as well as in the double-stranded (ds) and single-stranded (ss) reference molecules, was substituted with 2AP or...
62.
Specific inhibitors of HCV polymerase identified using an NS5B with lower affinity for template/primer substrate - McKercher, Ginette; Beaulieu, Pierre L.; Lamarre, Daniel; LaPlante, Steven; Lefebvre, Sylvain; Pellerin, Charles; Thauvette, Louise; Kukolj, George
The interaction of the hepatitis C virus (HCV) RNA-dependent RNA polymerase with RNA substrate is incompletely defined. We have characterized the activities of the HCV NS5B polymerase, modified by different deletions and affinity tags, with a routinely used homopolymeric substrate, and established apparent affinities of the various NS5B constructs both for the NTP and the template/primer substrates. We identified a uniquely tagged HCV NS5B RNA polymerase construct with a lower affinity (higher Km) than mature HCV NS5B for template/ primer substrate and highlighted the use of such a polymerase for the identification of inhibitors of NS5B activity, particularly inhibitors of...
63.
Synthesis of phosphorothioamidites derived from 3?-thio-3?-deoxythymidine and 3?-thio-2?,3?-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3?-S-phosphorothiolate linkage - Sabbagh, Ghalia; Fettes, Kevin J.; Gosain, Rajendra; O'Neil, Ian A.; Cosstick, Richard
The synthesis of N4-benzoyl-5?-O-dimethoxytrityl-2?,3?-dideoxy-3?-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5?-O-dimethoxytrityl-3?-deoxy-3?-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 8590% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 µmol reaction column, thus facilitating large scale syntheses required for mechanistic studies.
64.
A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase - Roovers, Martine; Wouters, Johan; Bujnicki, Janusz M.; Tricot, Catherine; Stalon, Victor; Grosjean, Henri; Droogmans, Louis
The modified nucleoside 1-methyladenosine (m1A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m1A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m1A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m1I57). In yeast, the formation of m1A58 is catalysed by the essential tRNA (m1A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is...
65.
Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome - Turan, Kadir; Mibayashi, Masaki; Sugiyama, Kenji; Saito, Shoko; Numajiri, Akiko; Nagata, Kyosuke
Mx proteins belong to the dynamin superfamily of high molecular weight GTPases and interfere with multiplication of a wide variety of viruses. Earlier studies show that nuclear mouse Mx1 and human MxA designed to be localized in the nucleus inhibit the transcription step of the influenza virus genome. Here we set a transient influenza virus transcription system using luciferase as a reporter gene and cells expressing the three RNA polymerase subunits, PB1, PB2 and PA, and NP. We used this reporter assay system and nuclear-localized MxA proteins to get clues for elucidating the anti-influenza virus activity of MxA. Nuclear-localized VP16-MxA...
66.
The sea urchin stemloop-binding protein: a maternally expressed protein that probably functions in expression of multiple classes of histone mRNA - Robertson, Anthony J.; Howard, Jason T.; Dominski, Zbigniew; Schnackenberg, Bradley J.; Sumerel, Jan L.; McCarthy, John J.; Coffman, James A.; Marzluff, William F.
Following the completion of oogenesis and oocyte maturation, histone mRNAs are synthesized and stored in the sea urchin egg pronucleus. Histone mRNAs are the only mRNAs that are not polyadenylated but instead end in a stemloop which has been conserved in evolution. The 3? end binds the stemloop-binding protein (SLBP), and SLBP is required for histone pre-mRNA processing as well as translation of the histone mRNAs. A cDNA encoding a 59 kDa sea urchin SLBP (suSLBP) has been cloned from an oocyte cDNA library. The suSLBP contains an RNA-binding domain that is similar to the RNA-binding domain found in SLBPs...
67.
Cassette-like variation of restriction enzyme genes in Escherichia coli C and relatives - Sibley, Marion H.; Raleigh, Elisabeth A.
A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species. We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli. This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there. To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C. Here we compare the 13.7 kb E.coli C sequence spanning the site of...
68.
Modification of de novo DNA methyltransferase 3a (Dnmt3a) by SUMO-1 modulates its interaction with histone deacetylases (HDACs) and its capacity to repress transcription - Ling, Yan; Sankpal, Umesh T.; Robertson, Andrea K.; McNally, James G.; Karpova, Tatiana; Robertson, Keith D.
The de novo DNA methyltransferase Dnmt3a is one of three mammalian DNA methyltransferases that has been shown to play crucial roles in embryonic development, genomic imprinting and transcriptional silencing. Despite its importance, very little is known about how the enzymatic activity and transcriptional repression functions of Dnmt3a are regulated. Here we show that Dnmt3a interacts with multiple components of the sumoylation machinery, namely the E2 sumo conjugating enzyme Ubc9 and the E3 sumo ligases PIAS1 and PIASx?, all of which are involved in conjugating the small ubiquitin-like modifier polypeptide, SUMO-1, to its target proteins. Dnmt3a is modified by SUMO-1 in...
69.
Guidelines for incorporating non-perfectly matched oligonucleotides into target-specific hybridization probes for a DNA microarray - Lee, Inhan; Dombkowski, Alan A.; Athey, Brian D.
Sequence-specific oligonucleotide probes play a crucial role in hybridization techniques including PCR, DNA microarray and RNA interference. Once the entire genome becomes the search space for target genes/genomic sequences, however, cross-hybridization to non-target sequences becomes a problem. Large gene families with significant similarity among family members, such as the P450s, are particularly problematic. Additionally, accurate single nucleotide polymorphism (SNP) detection depends on probes that can distinguish between nearly identical sequences. Conventional oligonucleotide probes that are perfectly matched to target genes/genomic sequences are often unsuitable in such cases. Carefully designed mismatches can be used to decrease cross-hybridization potential, but implementing all...
70.
Exon repetition: a major pathway for processing mRNA of some genes is allele-specific - Rigatti, Roberto; Jia, Jian-Hua; Samani, Nilesh J.; Eperon, Ian C.
Exon repetition describes the presence of tandemly repeated exons in mRNA in the absence of duplications in the genome. Its existence challenges our understanding of gene expression, because the linear organization of sequences in apparently normal genes must be subverted during RNA synthesis or processing. It is restricted to a small number of genes in some of which over half of the mRNA contains specific patterns of repetition. Although it is sometimes assumed to arise by trans-splicing, there is no evidence of this and the efficiency is very much higher than for examples of bona fide trans-splicing in mammals. Furthermore,...
71.
Design and synthesis of a photocleavable biotinylated nucleotide for DNA analysis by mass spectrometry - Bai, Xiaopeng; Kim, Sobin; Li, Zengmin; Turro, Nicholas J.; Ju, Jingyue
We report here the design, synthesis and evaluation of a novel photocleavable (PC) biotinylated nucleotide analog, dUTP-PC-Biotin, for DNA polymerase extension reaction to isolate DNA products for mass spectrometry (MS) analysis. This nucleotide analog has a biotin moiety attached to the 5-position of 2?-deoxyribouridine 5?-triphosphate via a photocleavable 2-nitrobenzyl linker. We have demonstrated that dUTP-PC-Biotin can be faithfully incorporated by the DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA polymerase extension reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. Therefore, the DNA extension fragments generated by using the dUTP-PC-Biotin can...
72.
Functional analysis of subcellular localization and proteinprotein interaction sequences in the essential DNA ligase I protein of fission yeast - Martin, Ina V.; MacNeill, Stuart A.
DNA ligase I (Lig I) has key roles in chromosomal DNA replication and repair in the eukaryotic cell nucleus. In the budding yeast Saccharomyces cerevisiae the Lig I enzyme Cdc9p is also required for mitochondrial DNA replication and repair. In this report, dual nuclearmitochondrial localization is demonstrated to be a property of the essential Lig I enzyme Cdc17 from the distantly related fission yeast Schizosaccharomyces pombe. Expression of nuclear and mitochondrial forms of Cdc17 from separate genes shows that, whereas expression of either protein alone is insufficient to restore viability to cells lacking endogenous Cdc17, co-expression restores full viability. In...
73.
Two mutations in the tetracycline repressor change the inducer anhydrotetracycline to a corepressor - Kamionka, Annette; Bogdanska-Urbaniak, Joanna; Scholz, Oliver; Hillen, Wolfgang
We report for the first time the in vitro characterization of a reverse tetracycline repressor (revTetR). The dimeric wild-type repressor (TetR) binds to tet operator tetO in the absence of the inducer anhydrotetracycline (atc) to confer tight repression. We have isolated the revTetR G96E L205S mutant, which, contrary to TetR, binds tetO only in the presence of atc. This reverse acting mutant was overproduced and purified. Effector and DNA binding properties were analyzed by EMSA and quantified by fluorescence titration and surface plasmon resonance. The association constant KA of revTetR for binding of [atcMg]+ is ?108 M1, four orders of...
74.
The major 5? determinant in stop codon read-through involves two adjacent adenines - Tork, Sanaa; Hatin, Isabelle; Rousset, Jean-Pierre; Fabret, Céline
The aim of this approach was to identify the major determinants, located at the 5? end of the stop codon, that modulate translational read-through in Saccharomyces cerevisiae. We developed a library of oligonucleotides degenerate at the six positions immediately upstream of the termination codon, cloned in the ADE2 reporter gene. Variations at these positions modulated translational read-through efficiency ?16-fold. The major effect was imposed by the two nucleotides immediately upstream of the stop codon. We showed that this effect was neither mediated by the last amino acid residues present in the polypeptide chain nor by the tRNA present in the...
75.
More active human L1 retrotransposons produce longer insertions - Farley, Alexander H.; Luning Prak, Eline T.; Kazazian, Haig H.
The vast majority of L1 insertions are 5? truncated and thus inactive. Yet, the mechanism of 5? truncation is unknown. To examine whether the frequency of L1 retrotransposition is directly correlated with the length of genomic L1 insertions, we used a cell culture assay to measure retrotransposition frequency and a PCR-based assay to measure L1 insertion length. We tested five full-length human L1 elements that retrotranspose at different frequencies: LRE3, L1RP, L1.3, L1.2A and L1.2B. Our data suggest that L1 insertion length correlates with L1 retrotransposition frequency for insertions >1 kb in length. For two elements, L1RP and L1.2A, we...
76.
Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control - Hsu, Chia-Ling; Chen, Ying-Shung; Tsai, Shih-Yin; Tu, Pei-Jung; Wang, Mei-Jung; Lin, Jing-Jer
Telomeres are the physical ends of eukaryotic chromosomes. They are important for maintaining the integrity of chromosomes and this function is mediated through a number of protein factors. In Saccharomyces cerevisiae, Cdc13p binds to telomeres and affects telomere maintenance, telomere position effects and cell cycle progression through G2/M phase. We identified four genes encoding Pol1p, Sir4p, Zds2p and Imp4p that interact with amino acids 1252 of Cdc13p using a yeast two-hybrid screening system. Interactions of these four proteins with Cdc13p were through direct proteinprotein interactions as judged by in vitro pull-down assays. Direct proteinprotein interactions were also observed between Pol1pImp4p,...
77.
Protein structure prediction using sparse dipolar coupling data - Qu, Youxing; Guo, Jun-tao; Olman, Victor; Xu, Ying
Residual dipolar coupling (RDC) represents one of the most exciting emerging NMR techniques for protein structure studies. However, solving a protein structure using RDC data alone is still a highly challenging problem. We report here a computer program, RDC-PROSPECT, for protein structure prediction based on a structural homolog or analog of the target protein in the Protein Data Bank (PDB), which best aligns with the 15N1H RDC data of the protein recorded in a single ordering medium. Since RDC-PROSPECT uses only RDC data and predicted secondary structure information, its performance is virtually independent of sequence similarity between a target protein...
78.
Statistical resynchronization and Bayesian detection of periodically expressed genes - Lu, Xin; Zhang, Wen; Qin, Zhaohui S.; Kwast, Kurt E.; Liu, Jun S.
We propose a periodicnormal mixture (PNM) model to fit transcription profiles of periodically expressed (PE) genes in cell cycle microarray experiments. The model leads to a principled statistical estimation procedure that produces more accurate estimates of the mean cell cycle length and the gene expression periodicity than existing heuristic approaches. A central component of the proposed procedure is the resynchronization of the observed transcription profile of each PE gene according to the PNM with estimated periodicity parameters. By using a two-component mixture-Beta model to approximate the PNM fitting residuals, we employ an empirical Bayes method to detect PE genes. We...
79.
Probing alternative foldings of the HIV-1 leader RNA by antisense oligonucleotide scanning arrays - Ooms, Marcel; Verhoef, Koen; Southern, Edwin; Huthoff, Hendrik; Berkhout, Ben
Scanning arrays of antisense DNA oligonucleotides provide a novel and systematic means to study structural features within an RNA molecule. We used this approach to probe the structure of the untranslated leader of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This 335 nt RNA encodes multiple important replication signals and adopts two mutually exclusive conformations. The poly(A) and the dimer initiation signal (DIS) sequences of the leader RNA are base-paired in the long-distance interaction (LDI) conformation, but both domains form distinct hairpins in the branched multiple hairpins (BMH) conformation. An RNA switch mechanism has been proposed to regulate...
80.
Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles - Miller, Victor M.; Gouvion, Cynthia M.; Davidson, Beverly L.; Paulson, Henry L.
Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimers disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M)...
61.
