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 81 - 100 de 59,293
81.
RNase H2 of Saccharomyces cerevisiae is a complex of three proteins - Jeong, Ho-Sang; Backlund, Peter S.; Chen, Hao-Chia; Karavanov, Alexander A.; Crouch, Robert J.
The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins (Rnh2Ap, Ydr279p and Ylr154p) that together are necessary and sufficient for RNase H2 activity. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when...
82.
Gene structure conservation aids similarity based gene prediction - Meyer, Irmtraud M.; Durbin, Richard
One of the primary tasks in deciphering the functional contents of a newly sequenced genome is the identification of its protein coding genes. Existing computational methods for gene prediction include ab initio methods which use the DNA sequence itself as the only source of information, comparative methods using multiple genomic sequences, and similarity based methods which employ the cDNA or protein sequences of related genes to aid the gene prediction. We present here an algorithm implemented in a computer program called Projector which combines comparative and similarity approaches. Projector employs similarity information at the genomic DNA level by directly using...
83.
Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase - Eastberg, Jennifer H.; Pelletier, John; Stoddard, Barry L.
T4 phage polynucleotide kinase (PNK) displays 5?-hydroxyl kinase, 3?-phosphatase and 2?,3?-cyclic phosphodiesterase activities. The enzyme phosphorylates the 5? hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5? ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases....
84.
Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts - Ichinoe, Akimasa; Behmanesh, Mehrdad; Tominaga, Yohei; Ushijima, Yasuhiro; Hirano, Seiki; Sakai, Yasunari; Tsuchimoto, Daisuke; Sakumi, Kunihiko; Wake, Norio; Nakabeppu, Yusaku
There are three types of mouse Mutyh mRNAs (type a, b and c) generated by alternative splicing, and type b mRNA is a major form among the three in most of the tissues examined. The level of type c mRNA is relatively high in brain. Type a and b mRNAs were expected to encode 57.7 kDa protein (MUTYH?), while type c mRNA had a partly different open reading frame encoding a 50.2 kDa protein (MUTYH?). An in vitro translation of type b and c mRNAs produced a 50 kDa MUTYH? and 47 kDa MUTYH?, respectively. MUTYH? and MUTYH? were detected...
85.
Crystal structure of the catalytic fragment of murine poly(ADP-ribose) polymerase-2 - Oliver, Antony W.; Amé, Jean-Christophe; Roe, S. Mark; Good, Valerie; de Murcia, Gilbert; Pearl, Laurence H.
Poly(ADP-ribose) polymerase-1 (PARP-1) has become an important pharmacological target in the treatment of cancer due to its cellular role as a DNA-strand break sensor, which leads in part to resistance to some existing chemo- and radiological treatments. Inhibitors have now been developed which prevent PARP-1 from synthesizing poly(ADP-ribose) in response to DNA-breaks and potentiate the cytotoxicity of DNA damaging agents. However, with the recent discoveries of PARP-2, which has a similar DNA-damage dependent catalytic activity, and additional members containing the PARP catalytic signature, the isoform selectivity and resultant pharmacological effects of existing inhibitors are brought into question. We present here...
86.
Automated correction of genome sequence errors - Gajer, Pawel; Schatz, Michael; Salzberg, Steven L.
By using information from an assembly of a genome, a new program called AutoEditor significantly improves base calling accuracy over that achieved by previous algorithms. This in turn improves the overall accuracy of genome sequences and facilitates the use of these sequences for polymorphism discovery. We describe the algorithm and its application in a large set of recent genome sequencing projects. The number of erroneous base calls in these projects was reduced by 80%. In an analysis of over one million corrections, we found that AutoEditor made just one error per 8828 corrections. By substantially increasing the accuracy of base...
87.
Surprising features of plastid ndhD transcripts: addition of non-encoded nucleotides and polysome association of mRNAs with an unedited start codon - Zandueta-Criado, Aitor; Bock, Ralph
RNA editing in higher plant plastids is a post- transcriptional RNA maturation process changing single cytidine nucleotides into uridine. In the ndhD transcript of tobacco and several other plant species, editing of an ACG codon to a standard AUG initiator codon is believed to be a prerequisite for translation. In order to test this assumption experimentally, we have analyzed the editing status of ndhD mRNA species in the process of translation. We show that unedited ndhD transcripts are also associated with polysomes in vivo, suggesting that they are translated. This surprising finding challenges the view that ACG to AUG editing...
88.
Premature termination codons enhance mRNA decapping in human cells - Couttet, P.; Grange, T.
Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance process that promotes selective degradation of imperfect messages containing premature translation termination codons (PTCs). In yeast, PTCs trigger both deadenylylation-independent mRNA decapping, thereby allowing their rapid degradation by a 5? to 3? exonuclease, and to a smaller extent accelerated deadenylylation. It is not clear to what extent this decay pathway is conserved in higher eukaryotes. We used a transcriptional pulse strategy relying on a tetracycline-regulated promoter to study the decay of a PTC- containing ?-globin mRNA in human cells. We show that a PTC destabilizes the mRNA and decreases its half-life from...
89.
Disruption of type III secretion in Salmonella enterica serovar Typhimurium by external guide sequences - McKinney, Jeffrey S.; Zhang, Haifeng; Kubori, Tomoko; Galán, Jorge E.; Altman, Sidney
The type III secretion system involved in Salmonella enterica serovar Typhimurium invasion of host cells has been disrupted using inducibly expressed oligonucleotide external guide sequences (EGSs) complementary to invB or invC mRNA. These EGSs direct single site cleavage in these mRNAs by endogenous RNase P, and their expression in Salmonella results in invC mRNA and InvC protein depletion, decreased type III secretion and interference with host cell invasion. Comparison of these effects with those from studies of Salmonella invB and invC mutants suggests that invB EGSs have polar effects on invC mRNA.
90.
2?-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo - Prakash, Thazha P.; Johnston, Joseph F.; Graham, Mark J.; Condon, Thomas P.; Manoharan, Muthiah
Synthesis and antisense activity of oligonucleotides modified with 2?-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2?-O-DMAOE) are described. The 2?-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide gapmers, with 2?-O-DMAOE- modified nucleoside residues at the ends and 2?-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. Gapmer oligonucleotides have one or two 2?-O-modified regions and a 2?-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2?-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression...
91.
Effects on RNAi of the tight structure, sequence and position of the targeted region - Yoshinari, Koichi; Miyagishi, Makoto; Taira, Kazunari
RNA interference (RNAi) is a gene-silencing phenomenon that involves the double-stranded RNA-mediated cleavage of mRNA, and small interfering RNAs (siRNAs) can cause RNAi in mammalian cells. There have been many attempts to clarify the mechanism of RNAi, but information about the relationship between the sequence and structure, in particular, a tight structure, of the target RNA and the activities of siRNAs are limited. In the present study, we examined this relationship by introducing the TAR element, which adopts a very stable secondary structure, at different positions within target RNAs. Our results suggested that the activities of siRNAs were affected by...
92.
Linker phosphoramidite reagents for the attachment of the first nucleoside to underivatized solid-phase supports - Pon, Richard T.; Yu, Shuyuan
New linker phosphoramidite reagents containing a cleavable 3?-ester linkage are used for attaching the first nucleoside to the surface of a solid- phase support. Inexpensive, underivatized amino supports, such as long chain alkylamine controlled-pore glass, can serve as universal supports. No modifications to phosphoramidite coupling conditions are required and, after synthesis, treatment with NH4OH releases the products with 3?-OH ends. No 3?-dephosphorylation is required. Phosphoramidite reagents containing a succinate and sulfonyl diethanol linkage between the nucleoside and phosphoramidite group are particularly advantageous and can be used to create both 3?-OH and 5?-phosphate ends on oligonucleotides. Reproducibility and quality of oligonucleotide...
93.
Characterization of the 5?-untranslated region of YB-1 mRNA and autoregulation of translation by YB-1 protein - Fukuda, Takao; Ashizuka, Megumi; Nakamura, Takanori; Shibahara, Kotaro; Maeda, Katsumasa; Izumi, Hiroto; Kohno, Kimitoshi; Kuwano, Michihiko; Uchiumi, Takeshi
The eukaryotic Y-box binding protein YB-1 is involved in various biological processes, including DNA repair, cell proliferation and the regulation of transcription and translation. YB-1 protein is abundant and expressed ubiquitously in human cells, functioning in cell proliferation and transformation. Its concentration is thought to be highly regulated at both the levels of transcription and translation. Therefore, we investigated whether or not the 5?-UTR of YB-1 mRNA affects the translation of YB-1 protein, thus influencing expression levels. Luciferase mRNA ligated to the YB-1 mRNA 5?-UTR was used as a reporter construct. Ligation of the full-length YB-1 5?-UTR (331 bases) enhanced...
94.
Catalytic and DNA-binding properties of the human Ogg1 DNA N-glycosylase/AP lyase: biochemical exploration of H270, Q315 and F319, three amino acids of the 8-oxoguanine-binding pocket - van der Kemp, Patricia Auffret; Charbonnier, Jean-Baptiste; Audebert, Marc; Boiteux, Serge
The human Ogg1 protein (hOgg1) is an antimutator DNA glycosylase/AP lyase that catalyzes the excision of 8-oxo-7,8-dihydroguanine (8-oxoG) and the incision of apurinic and apyrimidinic (AP) sites in DNA. In this study, we have investigated the functional role of H270, Q315 and F319, three amino acids that are located in the 8-oxoG-binding pocket of hOgg1. Wild-type and mutant hOgg1 proteins (H270A, H270R, H270L, Q315A and F319A) were purified to apparent homogeneity. The catalytic activities and the DNA-binding properties of the various hOgg1 mutants were compared to those of the wild-type. The results show that hOgg1 mutated at H270 (H270A and...
95.
Recent horizontal intron transfer to a chloroplast genome - Sheveleva, Elena V.; Hallick, Richard B.
Evidence is presented for the recent, horizontal transfer of a self-splicing, homing group II intron from a cyanobacteria to the chloroplast genome of Euglena myxocylindracea. The psbA gene of E.myxocylindracea was found to contain a single 2566 nt group II intron with a gene in domain 4 for a 575 amino acid maturase. The predicted secondary structure and tertiary interactions of the group II intron, as well as the derived maturase primary sequence, most closely resemble the homing intron of the cyanobacterium Calothrix and the rnl introns of Porphyra purpurea mitochondria, while being only distantly related to all other Euglena...
96.
Poly(ADP-ribose) polymerase (PARP-1) and p53 independently function in regulating double-strand break repair in primate cells - Süsse, Silke; Scholz, Claus-Jürgen; Bürkle, Alexander; Wiesmüller, Lisa
PARP-1 is rapidly activated by DNA strand breaks, which finally leads to the modulation of multiple protein activities in DNA replication, DNA repair and checkpoint control. PARP-1 may be involved in homologous recombination, and poly(ADP-ribosyl)ation of p53 represents one possible mechanism that activates p53 as a recombination surveillance factor. Here, we examined the influence of PARP-1 on homology-directed double-strand break (DSB) repair by use of a fluorescence- and I-SceI- meganuclease-based assay with either episomal or chromosomally integrated DNA substrates. Surprisingly, the transient expression of both full-length PARP-1 and of a dominant negative mutant, retaining the DNA-binding but lacking the catalytic...
97.
A novel concept for ligand attachment to oligonucleotides via a 2?-succinyl linker - Winkler, Johannes; Urban, Ernst; Losert, Doris; Wacheck, Volker; Pehamberger, Hubert; Noe, Christian R.
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2?-Amino-2?-deoxy-5?-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2? position. This compound was incorporated at the 3? end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotidefluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism...
98.
Role of the RNA polymerase ? subunits in CII-dependent activation of the bacteriophage ? pE promoter: identification of important residues and positioning of the ? C-terminal domains - Kedzierska, Barbara; Lee, David J.; W?grzyn, Grzegorz; Busby, Stephen J. W.; Thomas, Mark S.
The bacteriophage ? CII protein stimulates the activity of three phage promoters, pE, pI and paQ, upon binding to a site overlapping the 35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase ? subunit (?CTD) to demonstrate that one ?CTD binds near position 41 at pE, whilst the other ?CTD binds further upstream. The ?CTD bound near position 41 is oriented such that its 261 determinant is in close proximity to ?70. The location of ?CTD in CII-dependent complexes at...
99.
The Trypanosoma brucei spliced leader RNA and rRNA gene promoters have interchangeable TbSNAP50-binding elements - Schimanski, Bernd; Laufer, Gabriele; Gontcharova, Lilia; Günzl, Arthur
In the protist parasite Trypanosoma brucei, the small nuclear spliced leader (SL) RNA and the large rRNAs are key molecules for mRNA maturation and protein synthesis, respectively. The SL RNA gene (SLRNA) promoter recruits RNA polymerase II and consists of a bipartite upstream sequence element (USE) and an element close to the transcription initiation site. Here, we analyzed the distal part of the ribosomal (RRNA) promoter and identified two sequence blocks which, in reverse orientation, closely resemble the SLRNA USE by both sequence and spacing. A detailed mutational analysis revealed that the ribosomal (r)USE is essential for efficient RRNA transcription...
100.
On the interpretation of Raman spectra of 1-aminooxy-spermine/DNA complexes - Ruiz-Chica, A. J.; Medina, M. A.; Sánchez-Jiménez, F.; Ramírez, F. J.
By FTRaman spectroscopy, we have investigated the effect of 1-aminooxy-spermine (AOSPM) on aggregation and stability of calf-thymus DNA and selected oligonucleotide chains. AOSPM is able to mimic spermine in some macromolecular interactions, but is unable to substitute polyamines to maintain cell proliferation, suggesting pharmacological applications. Raman spectra of solutions containing AOSPM and either genomic DNA or two 15mer oligodeoxyribonucleotides, with GC or AT sequences, were recorded. Precipitation was observed for calf-thymus DNA, aggregated structures and appearance of several Z marker bands were observed for the 15mer GC sequence, and no macromolecular changes were detected for the 15mer AT sequence. Specific...
81.
