Nucleic Acids Research

The bHLH/Per-Arnt-Sim transcription factor SIM2 regulates muscle transcript myomesin2 via a novel, non-canonical E-box sequence.

Publication Date: 2008 May 14 PMID: 18480125
Authors: Woods, S. - Farrall, A. - Procko, C. - Whitelaw, M. L.
Journal: Nucleic Acids Res

Despite a growing number of descriptive studies that show Single-minded 2 (Sim2) is not only essential for murine survival, but also upregulated in colon, prostate and pancreatic tumours, there is a lack of direct target genes identified for this basic helix-loop-helix/PAS transcription factor. We have performed a set of microarray experiments aimed at identifying genes that are differentially regulated by SIM2, and successfully verified that the Myomesin2 (Myom2) gene is SIM2-responsive. Although SIM2 has been reported to be a transcription repressor, we find that SIM2 induces transcription of Myom2 and activates the Myom2 promoter sequence when co-expressed with the heterodimeric partner protein, ARNT1, in human embryonic kidney cells. Truncation and mutation of the Myom2 promoter sequence, combined with chromatin immunoprecipitation studies in cells, has lead to the delineation of a non-canonical E-box sequence 5'-AACGTG-3' that is bound by SIM2/ARNT1 heterodimers. Interestingly, in immortalized human myoblasts knock down of Sim2 results in increased levels of Myom2 RNA, suggesting that SIM2 is acting as a repressor in these cells and so its activity is likely to be highly context dependent. This is the first report of a direct SIM2/ARNT1 target gene with accompanying analysis of a functional response element.

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Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics.

Publication Date: 2008 May 14 PMID: 18480124
Authors: Yamashita, R. - Suzuki, Y. - Takeuchi, N. - Wakaguri, H. - Ueda, T. - Sugano, S. - Nakai, K.
Journal: Nucleic Acids Res

Although the knowledge accumulated on the transcriptional regulations of eukaryotes is significant, the knowledge on their translational regulations remains limited. Thus, we performed a comprehensive detection of terminal oligo-pyrimidine (TOP), which is one of the well-characterized cis-regulatory motifs for translational controls located immediately downstream of the transcriptional start sites of mRNAs. Utilizing our precise 5'-end information of the full-length cDNAs, we could screen 1645 candidate TOP genes by position specific matrix search. Among them, not only 75 out of 78 ribosomal protein genes but also eight previously identified non-ribosomal-protein TOP genes were included. We further experimentally validated the translational activities of 83 TOP candidate genes. Clear translational regulations exerted on the stimulation of 12-O-tetradecanoyl-1-phorbol-13-acetate for at least 41 of them was observed, indicating that there should be a few hundreds of human genes which are subjected to regulation at translation levels via TOPs. Our result suggests that TOP genes code not only formerly characterized ribosomal proteins and translation-related proteins but also a wider variety of proteins, such as lysosome-related proteins and metabolism-related proteins, playing pivotal roles in gene expression controls in the majority of cellular mRNAs.

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SerbGO: searching for the best GO tool.

Publication Date: 2008 May 14 PMID: 18480123
Authors: Mosquera, J. L. - Sanchez-Pla, A.
Journal: Nucleic Acids Res

In recent years, the scientific community has provided many tools to assist with pathway analysis. Some of these programs can be used to manage functional annotation of gene products, others are oriented to exploring and analyzing data sets and many allow both possibilities. Potential users of these tools are faced with the necessity to decide which of the existing programs are the most appropriate for their needs. SerbGO is a user-friendly web tool created to facilitate this task. It can be used (i) to search for specific functionalities and determine which applications provide them and (ii) to compare several applications on the basis of different types of functionalities. Iterating and combining both functionalities can easily lead to selecting an appropriate tool. Data required by SerbGO is either the desired capabilities within a defined Standard Functionalities Set or the list of the tools to be compared. The analysis performed carries out a cross-classification that produces an easily readable output with the list of tools that implement the capabilities demanded or a table with the categorization of the GO tools that one wishes to compare. SerbGO is freely available and does not require a login. It can be accessed either directly at our server (http://estbioinfo.stat.ub.es/apli/serbgo) or at the GO Consortium website (http://www.geneontology.org/GO.tools.microarray.shtml#serbgo).

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AsiDesigner: exon-based siRNA design server considering alternative splicing.

Publication Date: 2008 May 14 PMID: 18480122
Authors: Park, Y. K. - Park, S. M. - Choi, Y. C. - Lee, D. - Won, M. - Kim, Y. J.
Journal: Nucleic Acids Res

RNA interference (RNAi) with small interfering RNA (siRNA) has become a powerful tool in functional and medical genomic research through directed post-transcriptional gene silencing. In order to apply RNAi technique for eukaryotic organisms, where frequent alternative splicing results in diversification of mRNAs and finally of proteins, we need spliced mRNA isoform silencing to study the function of individual proteins. AsiDesigner is a web-based siRNA design software system, which provides siRNA design capability to account for alternative splicing for mRNA level gene silencing. It provides numerous novel functions including the designing of common siRNAs for the silencing of more than two mRNAs simultaneously, a scoring scheme to evaluate the performance of designed siRNAs by adopting currently known key design factors, a stepwise off-target searching with BLAST and FASTA algorithms and checking the folding secondary structure energy of siRNAs. To do this, we developed a novel algorithm to evaluate the common target region, where siRNAs can be designed to knockdown a specific mRNA isoform or more than two mRNA isoforms from a target gene simultaneously. The developed algorithm and the AsiDesigner were tested and validated as very effective throughout widely performed gene silencing experiments. It is expected that AsiDesigner will play an important role in functional genomics, drug discovery and other molecular biological research. AsiDesigner is freely accessible at http://sysbio.kribb.re.kr/AsiDesigner/.

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MADNet: microarray database network web server.

Publication Date: 2008 May 14 PMID: 18480121
Authors: Segota, I. - Bartonicek, N. - Vlahovicek, K.
Journal: Nucleic Acids Res

MADNet is a user-friendly data mining and visualization tool for rapid analysis of diverse high-throughput biological data such as microarray, phage display or even metagenome experiments. It presents biological information in the context of metabolic and signalling pathways, transcription factors and drug targets through minimal user input, consisting only of the file with the experimental data. These data are integrated with information stored in various biological databases such as NCBI nucleotide and protein databases, metabolic and signalling pathway databases (KEGG), transcription regulation (TRANSFAC(c)) and drug target database (DrugBank). MADNet is freely available for academic use at http://www.bioinfo.hr/madnet.

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GeneCAT--novel webtools that combine BLAST and co-expression analyses.

Publication Date: 2008 May 14 PMID: 18480120
Authors: Mutwil, M. - Obro, J. - Willats, W. G. - Persson, S.
Journal: Nucleic Acids Res

The gene co-expression analysis toolbox (GeneCAT) introduces several novel microarray data analyzing tools. First, the multigene co-expression analysis, combined with co-expressed gene networks, provides a more powerful data mining technique than standard, single-gene co-expression analysis. Second, the high-throughput Map-O-Matic tool matches co-expression pattern of multiple query genes to genes present in user-defined subdatabases, and can therefore be used for gene mapping in forward genetic screens. Third, Rosetta combines co-expression analysis with BLAST and can be used to find 'true' gene orthologs in the plant model organisms Arabidopsis thaliana and Hordeum vulgare (Barley). GeneCAT is equipped with expression data for the model plant A. thaliana, and first to introduce co-expression mining tools for the monocot Barley. GeneCAT is available at http://genecat.mpg.de.

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Terminal proteins of Streptomyces chromosome can target DNA into eukaryotic nuclei.

Publication Date: 2008 May 14 PMID: 18480119
Authors: Tsai, H. H. - Huang, C. H. - Lin, A. M. - Chen, C. W.
Journal: Nucleic Acids Res

Streptomyces species are highly abundant soil bacteria that possess linear chromosomes (and linear plasmids). The 5' ends of these molecules are covalently bound by terminal proteins (TPs), that are important for integrity and replication of the telomeres. There are at least two types of TPs, both of which contain a DNA-binding domain and a classical eukaryotic nuclear localization signal (NLS). Here we show that the NLS motifs on these TPs are highly efficient in targeting the proteins along with covalently bound plasmid DNA into the nuclei of human cells. The TP-mediated nuclear targeting resembles the inter-kingdom gene transfer mediated by Ti plasmids of Agrobacterium tumefaciens, in which a piece of the Ti plasmid DNA is targeted to the plant nuclei by a covalently bound NLS-containing protein. The discovery of the nuclear localization functions of the Streptomyces TPs not only suggests possible inter-kingdom gene exchanges between Streptomyces and eukaryotes in soil but also provides a novel strategy for gene delivery in humans and other eukaryotes.

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A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms.

Publication Date: 2008 May 14 PMID: 18480118
Authors: Leakey, T. I. - Zielinski, J. - Siegfried, R. N. - Siegel, E. R. - Fan, C. Y. - Cooney, C. A.
Journal: Nucleic Acids Res

DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites.

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High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.

Publication Date: 2008 May 13 PMID: 18477638
Authors: Mitra, S. - Shcherbakova, I. V. - Altman, R. B. - Brenowitz, M. - Laederach, A.
Journal: Nucleic Acids Res

The use of capillary electrophoresis with fluorescently labeled nucleic acids revolutionized DNA sequencing, effectively fueling the genomic revolution. We present an application of this technology for the high-throughput structural analysis of nucleic acids by chemical and enzymatic mapping ('footprinting'). We achieve the throughput and data quality necessary for genomic-scale structural analysis by combining fluorophore labeling of nucleic acids with novel quantitation algorithms. We implemented these algorithms in the CAFA (capillary automated footprinting analysis) open-source software that is downloadable gratis from https://simtk.org/home/cafa. The accuracy, throughput and reproducibility of CAFA analysis are demonstrated using hydroxyl radical footprinting of RNA. The versatility of CAFA is illustrated by dimethyl sulfate mapping of RNA secondary structure and DNase I mapping of a protein binding to a specific sequence of DNA. Our experimental and computational approach facilitates the acquisition of high-throughput chemical probing data for solution structural analysis of nucleic acids.

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The Predikin webserver: improved prediction of protein kinase peptide specificity using structural information.

Publication Date: 2008 May 13 PMID: 18477637
Authors: Saunders, N. F. - Kobe, B.
Journal: Nucleic Acids Res

The Predikin webserver allows users to predict substrates of protein kinases. The Predikin system is built from three components: a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. The Predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate-determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. The web interface provides user-friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. The server is available at http://predikin.biosci.uq.edu.au.

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KEGG Atlas mapping for global analysis of metabolic pathways.

Publication Date: 2008 May 13 PMID: 18477636
Authors: Okuda, S. - Yamada, T. - Hamajima, M. - Itoh, M. - Katayama, T. - Bork, P. - Goto, S. - Kanehisa, M.
Journal: Nucleic Acids Res

KEGG Atlas is a new graphical interface to the KEGG suite of databases, especially to the systems information in the PATHWAY and BRITE databases. It currently consists of a single global map and an associated viewer for metabolism, covering about 120 KEGG metabolic pathway maps and about 10 BRITE hierarchies. The viewer allows the user to navigate and zoom the global map under the Ajax technology. The mapping of high-throughput experimental data onto the global map is the main use of KEGG Atlas. In the global metabolism map, the node (circle) is a chemical compound and the edge (line) is a set of reactions linked to a set of KEGG Orthology (KO) entries for enzyme genes. Once gene identifiers in different organisms are converted to the K number identifiers in the KO system, corresponding line segments can be highlighted in the global map, allowing the user to view genome sequence data as organism-specific pathways, gene expression data as up- or down-regulated pathways, etc. Once chemical compounds are converted to the C number identifiers in KEGG, metabolomics data can also be displayed in the global map. KEGG Atlas is available at http://www.genome.jp/kegg/atlas/.

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Streptococcus pyogenes pSM19035 requires dynamic assembly of ATP-bound ParA and ParB on parS DNA during plasmid segregation.

Publication Date: 2008 May 13 PMID: 18477635
Authors: Pratto, F. - Cicek, A. - Weihofen, W. A. - Lurz, R. - Saenger, W. - Alonso, J. C.
Journal: Nucleic Acids Res

The accurate partitioning of Firmicute plasmid pSM19035 at cell division depends on ATP binding and hydrolysis by homodimeric ATPase delta(2) (ParA) and binding of omega(2) (ParB) to its cognate parS DNA. The 1.83 A resolution crystal structure of delta(2) in a complex with non-hydrolyzable ATPgammaS reveals a unique ParA dimer assembly that permits nucleotide exchange without requiring dissociation into monomers. In vitro, delta(2) had minimal ATPase activity in the absence of omega(2) and parS DNA. However, stoichiometric amounts of omega(2) and parS DNA stimulated the delta(2) ATPase activity and mediated plasmid pairing, whereas at high (4:1) omega(2) : delta(2) ratios, stimulation of the ATPase activity was reduced and delta(2) polymerized onto DNA. Stimulation of the delta(2) ATPase activity and its polymerization on DNA required ability of omega(2) to bind parS DNA and its N-terminus. In vivo experiments showed that delta(2) alone associated with the nucleoid, and in the presence of omega(2) and parS DNA, delta(2) oscillated between the nucleoid and the cell poles and formed spiral-like structures. Our studies indicate that the molar omega(2) : delta(2) ratio regulates the polymerization properties of (delta*ATP*Mg(2+))(2) on and depolymerization from parS DNA, thereby controlling the temporal and spatial segregation of pSM19035 before cell division.

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pFlexAna: detecting conformational changes in remotely related proteins.

Publication Date: 2008 May 13 PMID: 18477634
Authors: Nigham, A. - Tucker-Kellogg, L. - Mihalek, I. - Verma, C. - Hsu, D.
Journal: Nucleic Acids Res

The pFlexAna (protein flexibility analyzer) web server detects and displays conformational changes in remotely related proteins, without relying on sequence homology. To do so, it first applies a reliable statistical test to align core protein fragments that are structurally similar and then clusters these aligned fragment pairs into 'super-alignments', according to the similarity of geometric transformations that align them. The result is that the dominant conformational changes occur between the clusters, while the smaller conformational changes occur within a cluster. pFlexAna is available at http://bigbird.comp.nus.edu.sg/pfa2/.

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DNA conformations and their sequence preferences.

Publication Date: 2008 May 13 PMID: 18477633
Authors: Svozil, D. - Kalina, J. - Omelka, M. - Schneider, B.
Journal: Nucleic Acids Res

The geometry of the phosphodiester backbone was analyzed for 7739 dinucleotides from 447 selected crystal structures of naked and complexed DNA. Ten torsion angles of a near-dinucleotide unit have been studied by combining Fourier averaging and clustering. Besides the known variants of the A-, B- and Z-DNA forms, we have also identified combined A + B backbone-deformed conformers, e.g. with alpha/gamma switches, and a few conformers with a syn orientation of bases occurring e.g. in G-quadruplex structures. A plethora of A- and B-like conformers show a close relationship between the A- and B-form double helices. A comparison of the populations of the conformers occurring in naked and complexed DNA has revealed a significant broadening of the DNA conformational space in the complexes, but the conformers still remain within the limits defined by the A- and B- forms. Possible sequence preferences, important for sequence-dependent recognition, have been assessed for the main A and B conformers by means of statistical goodness-of-fit tests. The structural properties of the backbone in quadruplexes, junctions and histone-core particles are discussed in further detail.

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TargetRNA: a tool for predicting targets of small RNA action in bacteria.

Publication Date: 2008 May 13 PMID: 18477632
Authors: Tjaden, B.
Journal: Nucleic Acids Res

Many small RNA (sRNA) genes in bacteria act as posttranscriptional regulators of target messenger RNAs. Here, we present TargetRNA, a web tool for predicting mRNA targets of sRNA action in bacteria. TargetRNA takes as input a genomic sequence that may correspond to an sRNA gene. TargetRNA then uses a dynamic programming algorithm to search each annotated message in a specified genome for mRNAs that evince basepair-binding potential to the input sRNA sequence. Based on the calculated basepair-binding potential of each message with the given sRNA regulator, TargetRNA outputs a ranked list of candidate mRNA targets along with the predicted basepairing interaction of each target to the sRNA. The predictive performance of TargetRNA has been validated experimentally in several bacterial organisms. TargetRNA is freely available at http://snowwhite.wellesley.edu/targetRNA.

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AMIC@: All MIcroarray Clusterings @ once.

Publication Date: 2008 May 15 PMID: 18477631
Authors: Geraci, F. - Pellegrini, M. - Elena Renda, M.
Journal: Nucleic Acids Res

The AMIC@ Web Server offers a light-weight multi-method clustering engine for microarray gene-expression data. AMIC@ is a highly interactive tool that stresses user-friendliness and robustness by adopting AJAX technology, thus allowing an effective interleaved execution of different clustering algorithms and inspection of results. Among the salient features AMIC@ offers, there are: (i) automatic file format detection, (ii) suggestions on the number of clusters using a variant of the stability-based method of Tibshirani et al. (iii) intuitive visual inspection of the data via heatmaps and (iv) measurements of the clustering quality using cluster homogeneity. Large data sets can be processed efficiently by selecting algorithms (such as FPF-SB and k-Boost), specifically designed for this purpose. In case of very large data sets, the user can opt for a batch-mode use of the system by means of the Clustering wizard that runs all algorithms at once and delivers the results via email. AMIC@ is freely available and open to all users with no login requirement at the following URL http://bioalgo.iit.cnr.it/amica.

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Characterization of genome-wide p53-binding sites upon stress response.

Publication Date: 2008 May 12 PMID: 18474530
Authors: Smeenk, L. - van Heeringen, S. J. - Koeppel, M. - Driel, M. A. - Bartels, S. J. - Akkers, R. C. - Denissov, S. - Stunnenberg, H. G. - Lohrum, M.
Journal: Nucleic Acids Res

The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research.

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Gel-based oligonucleotide microarray approach to analyze protein-ssDNA binding specificity.

Publication Date: 2008 May 12 PMID: 18474529
Authors: Zasedateleva, O. A. - Mikheikin, A. L. - Turygin, A. Y. - Prokopenko, D. V. - Chudinov, A. V. - Belobritskaya, E. E. - Chechetkin, V. R. - Zasedatelev, A. S.
Journal: Nucleic Acids Res

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5'-{N}N(1)N(2)N(3)N(4)N(5)N(6){N}-3' consisted of a fixed hexamer motif N(1)N(2)N(3)N(4)N(5)N(6) in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase-ssDNA complexes with the temperature increasing from 0 degrees C to 50 degrees C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5'-NNG(A/T/C)GNN-3' with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.

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Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding.

Publication Date: 2008 May 12 PMID: 18474528
Authors: Roemer, S. C. - Adelman, J. - Churchill, M. E. - Edwards, D. P.
Journal: Nucleic Acids Res

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE binding, whereas the DNA architectural properties of HMGB are dispensable. Specific PRE DNA inhibited HMGB binding to the CTE, indicating that DNA and HMGB-CTE interactions are mutually exclusive. Exogenous CTE peptide increased PR-binding affinity for PRE as did deletion of the CTE. In a PR-binding site selection assay, A/T sequences flanking the PRE were enriched by HMGB, indicating that PR DNA-binding specificity is also altered by HMGB. We conclude that a transient HMGB-CTE interaction alters a repressive conformation of the flexible CTE enabling it to bind to preferred sequences flanking the PRE.

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LINE-1 methylation status of endogenous DNA double-strand breaks.

Publication Date: 2008 May 12 PMID: 18474527
Authors: Pornthanakasem, W. - Kongruttanachok, N. - Phuangphairoj, C. - Suyarnsestakorn, C. - Sanghangthum, T. - Oonsiri, S. - Ponyeam, W. - Thanasupawat, T. - Matangkasombut, O. - Mutirangura, A.
Journal: Nucleic Acids Res

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.

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Assessment of gene expression in many samples using vertical arrays.

Publication Date: 2008 May 12 PMID: 18474526
Authors: Risques, R. A. - Rondeau, G. - Judex, M. - McClelland, M. - Welsh, J.
Journal: Nucleic Acids Res

Microarrays and high-throughput sequencing methods can be used to measure the expression of thousands of genes in a biological sample in a few days, whereas PCR-based methods can be used to measure the expression of a few genes in thousands of samples in about the same amount of time. These methods become more costly as the number of biological samples increases or as the number of genes of interest increases, respectively, and these factors constrain experimental design. To address these issues, we introduced 'vertical arrays' in which RNA from each biological sample is converted into multiple, overlapping cDNA subsets and spotted on glass slides. These vertical arrays can be queried with single gene probes to assess the expression behavior in thousands of biological samples in a single hybridization reaction. The spotted subsets are less complex than the original RNA from which they derive, which improves signal-to-noise ratios. Here, we demonstrate the quantitative capabilities of vertical arrays, including the sensitivity and accuracy of the method and the number of subsets needed to achieve this accuracy for most expressed genes.

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pssRNAMiner: a plant short small RNA regulatory cascade analysis server.

Publication Date: 2008 May 12 PMID: 18474525
Authors: Dai, X. - Zhao, P. X.
Journal: Nucleic Acids Res

In plants, short RNAs including approximately 21-nt microRNA (miRNA) and 21-nt trans-acting siRNA (ta-siRNA) compose a 'miRNA --> ta-siRNA --> target gene' cascade pathway that regulates gene expression at the posttranscriptional level. In this cascade, biogenesis of ta-siRNA clusters requires 21-nt intervals (i.e. phasing) and miRNA (phase-initiator) cleavage sites on its TAS transcript. Here, we report a novel web server, pssRNAMiner, which is developed to identify both the clusters of phased small RNAs as well as the potential phase-initiator. To detect phased small RNA clusters, the pssRNAMiner maps input small RNAs against user-specified transcript/genomic sequences, and then identifies phased small RNA clusters by evaluating P-values of hypergeometric distribution. To identify potential phase-initiators, pssRNAMiner aligns input phase-initiators with transcripts of TAS candidates using the Smith-Waterman algorithm. Potential cleavage sites on TAS candidates are further identified from complementary regions by weighting the alignment expectation and its distance to detected phased small RNA clusters. The pssRNAMiner web server is freely available at http://bioinfo3.noble.org/pssRNAMiner/.

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MultiBind and MAPPIS: webservers for multiple alignment of protein 3D-binding sites and their interactions.

Publication Date: 2008 May 8 PMID: 18467424
Authors: Shulman-Peleg, A. - Shatsky, M. - Nussinov, R. - Wolfson, H. J.
Journal: Nucleic Acids Res

Analysis of protein-ligand complexes and recognition of spatially conserved physico-chemical properties is important for the prediction of binding and function. Here, we present two webservers for multiple alignment and recognition of binding patterns shared by a set of protein structures. The first webserver, MultiBind (http://bioinfo3d.cs.tau.ac.il/MultiBind), performs multiple alignment of protein binding sites. It recognizes the common spatial chemical binding patterns even in the absence of similarity of the sequences or the folds of the compared proteins. The input to the MultiBind server is a set of protein-binding sites defined by interactions with small molecules. The output is a detailed list of the shared physico-chemical binding site properties. The second webserver, MAPPIS (http://bioinfo3d.cs.tau.ac.il/MAPPIS), aims to analyze protein-protein interactions. It performs multiple alignment of protein-protein interfaces (PPIs), which are regions of interaction between two protein molecules. MAPPIS recognizes the spatially conserved physico-chemical interactions, which often involve energetically important hot-spot residues that are crucial for protein-protein associations. The input to the MAPPIS server is a set of protein-protein complexes. The output is a detailed list of the shared interaction properties of the interfaces.

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DNA binding of dinuclear iron(II) metallosupramolecular cylinders. DNA unwinding and sequence preference.

Publication Date: 2008 May 8 PMID: 18467423
Authors: Malina, J. - Hannon, M. J. - Brabec, V.
Journal: Nucleic Acids Res

[Fe(2)L(3)](4+) (L = C(25)H(20)N(4)) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe(2)L(3)](4+), we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine-pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe(2)(L-CF(3))(3)](4+) and [Fe(2)(L-Ph)(3)](4+) results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.

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PaLS: filtering common literature, biological terms and pathway information.

Publication Date: 2008 May 8 PMID: 18467422
Authors: Alibes, A. - Canada, A. - Diaz-Uriarte, R.
Journal: Nucleic Acids Res

Many biological experiments and their subsequent analysis yield lists of genes or proteins that can potentially be important to the prognosis or diagnosis of certain diseases (e.g. cancer). Nowadays, information about the function of those genes or proteins may be already gathered in some databases, but it is essential to understand if some of the members of those lists have a function in common or if they belong to the same metabolic pathway. To help researchers filter those genes or proteins that have such information in common, we have developed PaLS (pathway and literature strainer, http://pals.bioinfo.cnio.es). PaLS takes a list or a set of lists of gene or protein identifiers and shows which ones share certain descriptors. Four publicly available databases have been used for this purpose: PubMed, which links genes with those articles that make reference to them; Gene Ontology, an annotated ontology of terms related to the cellular component, biological process or molecular function where those genes or proteins are involved; KEGG pathways and Reactome pathways. Those descriptors among these four sources of information that are shared by more members of the list (or lists) are highlighted by PaLS.

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The Ontology Lookup Service: more data and better tools for controlled vocabulary queries.

Publication Date: 2008 May 8 PMID: 18467421
Authors: Cote, R. G. - Jones, P. - Martens, L. - Apweiler, R. - Hermjakob, H.
Journal: Nucleic Acids Res

The Ontology Lookup Service (OLS) (http://www.ebi.ac.uk/ols) provides interactive and programmatic interfaces to query, browse and navigate an ever increasing number of biomedical ontologies and controlled vocabularies. The volume of data available for querying has more than quadrupled since it went into production and OLS functionality has been integrated into several high-usage databases and data entry tools. Improvements have been made to both OLS query interfaces, based on user feedback and requirements, to improve usability and service interoperability and provide novel ways to perform queries.

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FFPred: an integrated feature-based function prediction server for vertebrate proteomes.

Publication Date: 2008 May 7 PMID: 18463141
Authors: Lobley, A. E. - Nugent, T. - Orengo, C. A. - Jones, D. T.
Journal: Nucleic Acids Res

One of the challenges of the post-genomic era is to provide accurate function annotations for large volumes of data resulting from genome sequencing projects. Most function prediction servers utilize methods that transfer existing database annotations between orthologous sequences. In contrast, there are few methods that are independent of homology and can annotate distant and orphan protein sequences. The FFPred server adopts a machine-learning approach to perform function prediction in protein feature space using feature characteristics predicted from amino acid sequence. The features are scanned against a library of support vector machines representing over 300 Gene Ontology (GO) classes and probabilistic confidence scores returned for each annotation term. The GO term library has been modelled on human protein annotations; however, benchmark performance testing showed robust performance across higher eukaryotes. FFPred offers important advantages over traditional function prediction servers in its ability to annotate distant homologues and orphan protein sequences, and achieves greater coverage and classification accuracy than other feature-based prediction servers. A user may upload an amino acid and receive annotation predictions via email. Feature information is provided as easy to interpret graphics displayed on the sequence of interest, allowing for back-interpretation of the associations between features and function classes.

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NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11.

Publication Date: 2008 May 7 PMID: 18463140
Authors: Lundegaard, C. - Lamberth, K. - Harndahl, M. - Buus, S. - Lund, O. - Nielsen, M.
Journal: Nucleic Acids Res

NetMHC-3.0 is trained on a large number of quantitative peptide data using both affinity data from the Immune Epitope Database and Analysis Resource (IEDB) and elution data from SYFPEITHI. The method generates high-accuracy predictions of major histocompatibility complex (MHC): peptide binding. The predictions are based on artificial neural networks trained on data from 55 MHC alleles (43 Human and 12 non-human), and position-specific scoring matrices (PSSMs) for additional 67 HLA alleles. As only the MHC class I prediction server is available, predictions are possible for peptides of length 8-11 for all 122 alleles. artificial neural network predictions are given as actual IC(50) values whereas PSSM predictions are given as a log-odds likelihood scores. The output is optionally available as download for easy post-processing. The training method underlying the server is the best available, and has been used to predict possible MHC-binding peptides in a series of pathogen viral proteomes including SARS, Influenza and HIV, resulting in an average of 75-80% confirmed MHC binders. Here, the performance is further validated and benchmarked using a large set of newly published affinity data, non-redundant to the training set. The server is free of use and available at: http://www.cbs.dtu.dk/services/NetMHC.

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Molecular basis for the lack of enantioselectivity of human 3-phosphoglycerate kinase.

Publication Date: 2008 May 7 PMID: 18463139
Authors: Gondeau, C. - Chaloin, L. - Lallemand, P. - Roy, B. - Perigaud, C. - Barman, T. - Varga, A. - Vas, M. - Lionne, C. - Arold, S. T.
Journal: Nucleic Acids Res

Non-natural l-nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l-nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l-nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the interaction between PGK and the bases. This, combined with the dispensability of hydrogen bonds to the sugar moiety, and ionic interactions with the phosphate groups, results in the positioning of different nucleotides so to expose their diphosphate group in a position competent for catalysis. Since the third phosphorylation step is often rate limiting, our results are expected to alleviate in silico tailoring of l-type prodrugs to assure their efficient metabolic processing.

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Microarray retriever: a web-based tool for searching and large scale retrieval of public microarray data.

Publication Date: 2008 May 7 PMID: 18463138
Authors: Ivliev, A. E. - 't Hoen, P. A. - Villerius, M. P. - den Dunnen, J. T. - Brandt, B. W.
Journal: Nucleic Acids Res

The major public microarray repositories Gene Expression Omnibus and ArrayExpress are growing rapidly. This enables meta-analysis studies, in which expression data from multiple individual studies are combined. To facilitate these types of studies, we developed Microarray Retriever for searching and retrieval of data from GEO and ArrayExpress. The tool allows access to the two repositories simultaneously, to search in the repositories using complex queries, to retrieve microarray data for published articles and to download data in one structured archive. The tool is available on the web at: http://www.lgtc.nl/MaRe/

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SEQATOMS: a web tool for identifying missing regions in PDB in sequence context.

Publication Date: 2008 May 7 PMID: 18463137
Authors: Brandt, B. W. - Heringa, J. - Leunissen, J. A.
Journal: Nucleic Acids Res

With over 46 000 proteins, the Protein Data Bank (PDB) is the most important database with structural information of biological macromolecules. PDB files contain sequence and coordinate information. Residues present in the sequence can be absent from the coordinate section, which means their position in space is unknown. Similarity searches are routinely carried out against sequences taken from PDB SEQRES. However, there no distinction is made between residues that have a known or unknown position in the 3D protein structure. We present a FASTA sequence database that is produced by combining the sequence and coordinate information. All residues absent from the PDB coordinate section are masked with lower-case letters, thereby providing a view of these residues in the context of the entire protein sequence, which facilitates inspecting 'missing' regions. We also provide a masked version of the CATH domain database. A user-friendly BLAST interface is available for similarity searching. In contrast to standard (stand-alone) BLAST output, which only contains upper-case letters, our output retains the lower-case letters of the masked regions. Thus, our server can be used to perform BLAST searching case-sensitively. Here, we have applied it to the study of missing regions in their sequence context. SEQATOMS is available at http://www.bioinformatics.nl/tools/seqatoms/.

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The Jpred 3 secondary structure prediction server.

Publication Date: 2008 May 7 PMID: 18463136
Authors: Cole, C. - Barber, J. D. - Barton, G. J.
Journal: Nucleic Acids Res

Jpred (http://www.compbio.dundee.ac.uk/jpred) is a secondary structure prediction server powered by the Jnet algorithm. Jpred performs over 1000 predictions per week for users in more than 50 countries. The recently updated Jnet algorithm provides a three-state (alpha-helix, beta-strand and coil) prediction of secondary structure at an accuracy of 81.5%. Given either a single protein sequence or a multiple sequence alignment, Jpred derives alignment profiles from which predictions of secondary structure and solvent accessibility are made. The predictions are presented as coloured HTML, plain text, PostScript, PDF and via the Jalview alignment editor to allow flexibility in viewing and applying the data. The new Jpred 3 server includes significant usability improvements that include clearer feedback of the progress or failure of submitted requests. Functional improvements include batch submission of sequences, summary results via email and updates to the search databases. A new software pipeline will enable Jnet/Jpred to continue to be updated in sync with major updates to SCOP and UniProt and so ensures that Jpred 3 will maintain high-accuracy predictions.

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OREST: the online resource for EST analysis.

Publication Date: 2008 May 7 PMID: 18463135
Authors: Waegele, B. - Schmidt, T. - Mewes, H. W. - Ruepp, A.
Journal: Nucleic Acids Res

The generation of expressed sequence tag (EST) libraries offers an affordable approach to investigate organisms, if no genome sequence is available. OREST (http://mips.gsf.de/genre/proj/orest/index.html) is a server-based EST analysis pipeline, which allows the rapid analysis of large amounts of ESTs or cDNAs from mammalia and fungi. In order to assign the ESTs to genes or proteins OREST maps DNA sequences to reference datasets of gene products and in a second step to complete genome sequences. Mapping against genome sequences recovers additional 13% of EST data, which otherwise would escape further analysis. To enable functional analysis of the datasets, ESTs are functionally annotated using the hierarchical FunCat annotation scheme as well as GO annotation terms. OREST also allows to predict the association of gene products and diseases by Morbid Map (OMIM) classification. A statistical analysis of the results of the dataset is possible with the included PROMPT software, which provides information about enrichment and depletion of functional and disease annotation terms. OREST was successfully applied for the identification and functional characterization of more than 3000 EST sequences of the common marmoset monkey (Callithrix jacchus) as part of an international collaboration.

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TRE-dependent transcription activation by JDP2-CHOP10 association.

Publication Date: 2008 May 7 PMID: 18463134
Authors: Weidenfeld-Baranboim, K. - Bitton-Worms, K. - Aronheim, A.
Journal: Nucleic Acids Res

The c-Jun dimerization protein 2, JDP2, is a member of the activating protein 1 (AP-1) family of transcription factors. Overexpression of JDP2 has been shown to result in repression of AP-1-dependent transcription and inhibition of cellular transformation. Other studies suggested that JDP2 may function as an oncogene. Here we describe the identification of CHOP10, a member of the CCAAT enhancer binding proteins, as a protein associating with JDP2. In contrast to the inhibition of transcription by JDP2, JDP2-CHOP complex strongly enhances transcription from promoters containing TPA response elements (TRE), but not from those containing cyclic AMP response elements (CRE). The association between JDP2 and CHOP10 involves the leucine zipper motifs of both proteins, whereas, the basic domain of CHOP10 contributes to the association of the JDP2-CHOP10 complex with the DNA. DNA binding of JDP2-CHOP complex is observed both in vitro and in vivo. Finally, overexpression of JDP2 results in increased cell viability following ER stress and counteracts CHOP10 pro-apoptotic activity. JDP2 expression may determine the threshold for cell sensitivity to ER stress. This is the first report describing TRE-dependent activation of transcription by JDP2 and thus may provide an explanation for the as yet unexplored oncogenic properties of JDP2.

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RAPIDO: a web server for the alignment of protein structures in the presence of conformational changes.

Publication Date: 2008 May 6 PMID: 18460546
Authors: Mosca, R. - Schneider, T. R.
Journal: Nucleic Acids Res

Rapid alignment of proteins in terms of domains (RAPIDO) is a web server for the 3D alignment of crystal structures of different protein molecules in the presence of conformational change. The structural alignment algorithm identifies groups of equivalent atoms whose interatomic distances are constant (within a defined tolerance) in the two structures being compared and considers these groups of atoms as rigid bodies. In addition to the functionalities provided by existing tools, RAPIDO can identify structurally equivalent regions also when these consist of fragments that are distant in terms of sequence and separated by other movable domains. Furthermore, RAPIDO takes the variation in the reliability of atomic coordinates into account in the comparison of distances between equivalent atoms by employing weighting-functions based on the refined B-values. The regions identified as equivalent by RAPIDO furnish reliable sets of residues for the superposition of the two structures for subsequent detailed analysis. The RAPIDO server, with related documentation, is available at http://webapps.embl-hamburg.de/rapido.

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Selection of a novel class of RNA-RNA interaction motifs based on the ligase ribozyme with defined modular architecture.

Publication Date: 2008 May 6 PMID: 18460545
Authors: Ohuchi, S. P. - Ikawa, Y. - Nakamura, Y.
Journal: Nucleic Acids Res

To develop molecular tools for the detection and control of RNA molecules whose functions rely on their 3D structures, we have devised a selection system to isolate novel RNA motifs that interact with a target RNA structure within a given structural context. In this system, a GAAA tetraloop and its specific receptor motif (11-ntR) from an artificial RNA ligase ribozyme with modular architecture (the DSL ribozyme) were replaced with a target structure and random sequence, respectively. Motifs recognizing the target structure can be identified by in vitro selection based on ribozyme activity. A model selection targeting GAAA-loop successfully identified motifs previously known as GAAA-loop receptors. In addition, a new selection targeting a C-loop motif also generated novel motifs that interact with this structure. Biochemical analysis of one of the C-loop receptor motifs revealed that it could also function as an independent structural unit.

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GraphWeb: mining heterogeneous biological networks for gene modules with functional significance.

Publication Date: 2008 May 6 PMID: 18460544
Authors: Reimand, J. - Tooming, L. - Peterson, H. - Adler, P. - Vilo, J.
Journal: Nucleic Acids Res

Deciphering heterogeneous cellular networks with embedded modules is a great challenge of current systems biology. Experimental and computational studies construct complex networks of molecules that describe various aspects of the cell such as transcriptional regulation, protein interactions and metabolism. Groups of interacting genes and proteins reflect network modules that potentially share regulatory mechanisms and relate to common function. Here, we present GraphWeb, a public web server for biological network analysis and module discovery. GraphWeb provides methods to: (1) integrate heterogeneous and multispecies data for constructing directed and undirected, weighted and unweighted networks; (ii) discover network modules using a variety of algorithms and topological filters and (iii) interpret modules using functional knowledge of the Gene Ontology and pathways, as well as regulatory features such as binding motifs and microRNA targets. GraphWeb is designed to analyse individual or multiple merged networks, search for conserved features across multiple species, mine large biological networks for smaller modules, discover novel candidates and connections for known pathways and compare results of high-throughput datasets. The GraphWeb is available at http://biit.cs.ut.ee/graphweb/.

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ProfCom: a web tool for profiling the complex functionality of gene groups identified from high-throughput data.

Publication Date: 2008 May 6 PMID: 18460543
Authors: Antonov, A. V. - Schmidt, T. - Wang, Y. - Mewes, H. W.
Journal: Nucleic Acids Res

ProfCom is a web-based tool for the functional interpretation of a gene list that was identified to be related by experiments. A trait which makes ProfCom a unique tool is an ability to profile enrichments of not only available Gene Ontology (GO) terms but also of 'complex functions'. A 'Complex function' is constructed as Boolean combination of available GO terms. The complex functions inferred by ProfCom are more specific in comparison to single terms and describe more accurately the functional role of genes. ProfCom provides a user friendly dialog-driven web page submission available for several model organisms and supports most available gene identifiers. In addition, the web service interface allows the submission of any kind of annotation data. ProfCom is freely available at http://webclu.bio.wzw.tum.de/profcom/.

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Cooperation between EZH2, NSPc1-mediated histone H2A ubiquitination and Dnmt1 in HOX gene silencing.

Publication Date: 2008 May 6 PMID: 18460542
Authors: Wu, X. - Gong, Y. - Yue, J. - Qiang, B. - Yuan, J. - Peng, X.
Journal: Nucleic Acids Res

An intricate interplay between DNA methylation and polycomb-mediated gene silencing has been highlighted recently. Here we provided evidence that Nervous System Polycomb 1 (NSPc1), a BMI1 homologous polycomb protein, plays important roles in promoting H2A ubiquitination and cooperates with DNA methylation in HOX gene silencing. We showed that NSPc1 stimulates H2A ubiquitination in vivo and in vitro through direct interaction with both RING2 and H2A. RT-PCR analysis revealed that loss of NSPc1, EZH2 or DNA methyltransferase 1 (Dnmt1), or inhibition of DNA methylation in HeLa cells de-represses the expression of HOXA7. Chromatin immunoprecipitation (ChIP) assays demonstrated that NSPc1, EZH2 and Dnmt1 bind to the promoter of HOXA7, which is frequently hypermethylated in tumors. Knockdown of NSPc1 results in significant reduction of H2A ubiquitination and DNA demethylation as well as Dnmt1 dissociation in the HOXA7 promoter. Meanwhile Dnmt1 deficiency affects NSPc1 recruitment and H2A ubiquitination, whereas on both cases EZH2-mediated H3K27 trimethylation remains unaffected. When EZH2 was depleted, however, NSPc1 and Dnmt1 enrichment was abolished concomitant with local reduction of H3K27 trimethylation, H2A ubiquitination and DNA methylation. Taken together, our findings indicated that NSPc1-mediated H2A ubiquitination and DNA methylation, both being directed by EZH2, are interdependent in long-term target gene silencing within cancer cells.

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Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses.

Publication Date: 2008 May 2 PMID: 18456708
Authors: Orlowski, J. - Bujnicki, J. M.
Journal: Nucleic Acids Res

For a very long time, Type II restriction enzymes (REases) have been a paradigm of ORFans: proteins with no detectable similarity to each other and to any other protein in the database, despite common cellular and biochemical function. Crystallographic analyses published until January 2008 provided high-resolution structures for only 28 of 1637 Type II REase sequences available in the Restriction Enzyme database (REBASE). Among these structures, all but two possess catalytic domains with the common PD-(D/E)XK nuclease fold. Two structures are unrelated to the others: R.BfiI exhibits the phospholipase D (PLD) fold, while R.PabI has a new fold termed 'half-pipe'. Thus far, bioinformatic studies supported by site-directed mutagenesis have extended the number of tentatively assigned REase folds to five (now including also GIY-YIG and HNH folds identified earlier in homing endonucleases) and provided structural predictions for dozens of REase sequences without experimentally solved structures. Here, we present a comprehensive study of all Type II REase sequences available in REBASE together with their homologs detectable in the nonredundant and environmental samples databases at the NCBI. We present the summary and critical evaluation of structural assignments and predictions reported earlier, new classification of all REase sequences into families, domain architecture analysis and new predictions of three-dimensional folds. Among 289 experimentally characterized (not putative) Type II REases, whose apparently full-length sequences are available in REBASE, we assign 199 (69%) to contain the PD-(D/E)XK domain. The HNH domain is the second most common, with 24 (8%) members. When putative REases are taken into account, the fraction of PD-(D/E)XK and HNH folds changes to 48% and 30%, respectively. Fifty-six characterized (and 521 predicted) REases remain unassigned to any of the five REase folds identified so far, and may exhibit new architectures. These enzymes are proposed as the most interesting targets for structure determination by high-resolution experimental methods. Our analysis provides the first comprehensive map of sequence-structure relationships among Type II REases and will help to focus the efforts of structural and functional genomics of this large and biotechnologically important class of enzymes.

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Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments.

Publication Date: 2008 May 2 PMID: 18456707
Authors: Yokoyama, T. - Suzuki, T.
Journal: Nucleic Acids Res

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Delta7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.

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OGtree: a tool for creating genome trees of prokaryotes based on overlapping genes.

Publication Date: 2008 May 2 PMID: 18456706
Authors: Jiang, L. W. - Lin, K. L. - Lu, C. L.
Journal: Nucleic Acids Res

OGtree is a web-based tool for constructing genome trees of prokaryotic species based on a measure of combining overlapping-gene content and overlapping-gene order in their whole genomes. The overlapping genes (OGs) are defined as adjacent genes whose coding sequences overlap partially or entirely. In fact, OGs are ubiquitous in microbial genomes and more conserved between species than non-OGs. Based on these properties, it has been suggested that OGs can serve as better phylogenetic characters than non-OGs for reconstructing the evolutionary relationships among microbial genomes. OGtree takes the accession numbers of prokaryotic genomes as its input. It then downloads their complete genomes from the National Centre for Biotechnology Information and identifies OGs in each genome and their orthologous OGs in other genomes. Next, OGtree computes an overlapping-gene distance between each pair of input genomes based on a combination of their OG content and orthologous OG order. Finally, it utilizes distance-based methods of building tree to reconstruct the genome trees of input prokaryotic genomes according to their pairwise OG distance. OGtree is available online at http://bioalgorithm.life.nctu.edu.tw/OGtree/.

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Purine twisted-intercalating nucleic acids: a new class of anti-gene molecules resistant to potassium-induced aggregation.

Publication Date: 2008 May 2 PMID: 18456705
Authors: Paramasivam, M. - Cogoi, S. - Filichev, V. V. - Bomholt, N. - Pedersen, E. B. - Xodo, L. E.
Journal: Nucleic Acids Res

Sequence-specific targeting of genomic DNA by triplex-forming oligonucleotides (TFOs) is a promising strategy to modulate in vivo gene expression. Triplex formation involving G-rich oligonucleotides as third strand is, however, strongly inhibited by potassium-induced TFO self-association into G-quartet structures. We report here that G-rich TFOs with bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)-phenylmethyl] glycerol (called twisted intercalating nucleic acids, TINA) show a much lower tendency to aggregate in potassium than wild-type analogues do. We designed purine-motif TINA-TFOs for binding to a regulatory polypurine-polypyrimidine (pur/pyr) motif present in the promoter of the KRAS proto-oncogene. The binding of TINA-TFOs to the KRAS target has been analysed by electrophoresis mobility shift assays and DNase I footprinting experiments. We discovered that in the presence of potassium the wild-type TFOs did not bind to the KRAS target, differently from the TINA analogues, whose binding was observed up to 140 mM KCl. The designed TINA-TFOs were found to abrogate the formation of a DNA-protein complex at the pur/pyr site and to down-regulate the transcription of CAT driven by the murine KRAS promoter. Molecular modelling of the DNA/TINA-TFO triplexes are also reported. This study provides a new and promising approach to create TFOs to target in vivo the genome.

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Structural and functional insights into human Tudor-SN, a key component linking RNA interference and editing.

Publication Date: 2008 May 3 PMID: 18453631
Authors: Li, C. L. - Yang, W. Z. - Chen, Y. P. - Yuan, H. S.
Journal: Nucleic Acids Res

Human Tudor-SN is involved in the degradation of hyper-edited inosine-containing microRNA precursors, thus linking the pathways of RNA interference and editing. Tudor-SN contains four tandem repeats of staphylococcal nuclease-like domains (SN1-SN4) followed by a tudor and C-terminal SN domain (SN5). Here, we showed that Tudor-SN requires tandem repeats of SN domains for its RNA binding and cleavage activity. The crystal structure of a 64-kD truncated form of human Tudor-SN further shows that the four domains, SN3, SN4, tudor and SN5, assemble into a crescent-shaped structure. A concave basic surface formed jointly by SN3 and SN4 domains is likely involved in RNA binding, where citrate ions are bound at the putative RNase active sites. Additional modeling studies provide a structural basis for Tudor-SN's preference in cleaving RNA containing multiple I.U wobble-paired sequences. Collectively, these results suggest that tandem repeats of SN domains in Tudor-SN function as a clamp to capture RNA substrates.

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Crystal structure of trioxacarcin A covalently bound to DNA.

Publication Date: 2008 May 3 PMID: 18453630
Authors: Pfoh, R. - Laatsch, H. - Sheldrick, G. M.
Journal: Nucleic Acids Res

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule 'caught in the act' after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 A resolution by MAD phasing employing brominated oligonucleotides. The DNA-drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3'-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.

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Spatial effects on the speed and reliability of protein-DNA search.

Publication Date: 2008 May 3 PMID: 18453629
Authors: Wunderlich, Z. - Mirny, L. A.
Journal: Nucleic Acids Res

Strong experimental and theoretical evidence shows that transcription factors (TFs) and other specific DNA-binding proteins find their sites using a two-mode search: alternating between three-dimensional (3D) diffusion through the cell and one-dimensional (1D) sliding along the DNA. We show that, due to the 1D component of the search process, the search time of a TF can depend on the initial position of the TF. We formalize this effect by discriminating between two types of searches: global and local. Using analytical calculations and simulations, we estimate how close a TF and binding site need to be to make a local search likely. We then use our model to interpret the wide range of experimental measurements of this parameter. We also show that local and global searches differ significantly in average search time and the variability of search time. These results lead to a number of biological implications, including suggestions of how prokaryotes achieve rapid gene regulation and the relationship between the search mechanism and noise in gene expression. Lastly, we propose a number of experiments to verify the existence and quantify the extent of spatial effects on the TF search process in prokaryotes.

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ConTra: a promoter alignment analysis tool for identification of transcription factor binding sites across species.

Publication Date: 2008 May 3 PMID: 18453628
Authors: Hooghe, B. - Hulpiau, P. - van Roy, F. - De Bleser, P.
Journal: Nucleic Acids Res

Transcription factors (TFs) are key components in signaling pathways, and the presence of their binding sites in the promoter regions of DNA is essential for their regulation of the expression of the corresponding genes. Orthologous promoter sequences are commonly used to increase the specificity with which potentially functional transcription factor binding sites (TFBSs) are recognized and to detect possibly important similarities or differences between the different species. The ConTra (conserved TFBSs) web server provides the biologist at the bench with a user-friendly tool to interactively visualize TFBSs predicted using either TransFac (1) or JASPAR (2) position weight matrix libraries, on a promoter alignment of choice. The visualization can be preceded by a simple scoring analysis to explore which TFs are the most likely to bind to the promoter of interest. The ConTra web server is available at http://bioit.dmbr.ugent.be/ConTra/index.php.

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Human and mouse introns are linked to the same processes and functions through each genome's most frequent non-conserved motifs.

Publication Date: 2008 May 1 PMID: 18450818
Authors: Tsirigos, A. - Rigoutsos, I.
Journal: Nucleic Acids Res

We identified the most frequent, variable-length DNA sequence motifs in the human and mouse genomes and sub-selected those with multiple recurrences in the intergenic and intronic regions and at least one additional exonic instance in the corresponding genome. We discovered that these motifs have virtually no overlap with intronic sequences that are conserved between human and mouse, and thus are genome-specific. Moreover, we found that these motifs span a substantial fraction of previously uncharacterized human and mouse intronic space. Surprisingly, we found that these genome-specific motifs are over-represented in the introns of genes belonging to the same biological processes and molecular functions in both the human and mouse genomes even though the underlying sequences are not conserved between the two genomes. In fact, the processes and functions that are linked to these genome-specific sequence-motifs are distinct from the processes and functions which are associated with intronic regions that are conserved between human and mouse. The findings show that intronic regions from different genomes are linked to the same processes and functions in the absence of underlying sequence conservation. We highlight the ramifications of this observation with a concrete example that involves the microsatellite instability gene MLH1.

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Chemical mapping of cytosines enzymatically flipped out of the DNA helix.

Publication Date: 2008 May 1 PMID: 18450817
Authors: Daujotyte, D. - Liutkeviciute, Z. - Tamulaitis, G. - Klimasauskas, S.
Journal: Nucleic Acids Res

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein-DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein-DNA complexes.

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Sequence-specific DNA cleavage mediated by bipyridine polyamide conjugates.

Publication Date: 2008 May 1 PMID: 18450816
Authors: Simon, P. - Cannata, F. - Perrouault, L. - Halby, L. - Concordet, J. P. - Boutorine, A. - Ryabinin, V. - Sinyakov, A. - Giovannangeli, C.
Journal: Nucleic Acids Res

The design of molecules that damage a selected DNA sequence provides a formidable opportunity for basic and applied biology. For example, such molecules offer new prospects for controlled manipulation of the genome. The conjugation of DNA-code reading molecules such as polyamides to reagents that induce DNA damages provides an approach to reach this goal. In this work, we showed that a bipyridine conjugate of polyamides was able to induce sequence-specific DNA breaks in cells. We synthesized compounds based on two polyamide parts linked to bipyridine at different positions. Bipyridine conjugates of polyamides were found to have a high affinity for the DNA target and one of them produced a specific and high-yield cleavage in vitro and in cultured cells. The bipyridine conjugate studied here, also presents cell penetrating properties since it is active when directly added to cell culture medium. Harnessing DNA damaging molecules such as bipyridine to predetermined genomic sites, as achieved here, provides an attractive strategy for targeted genome modification and DNA repair studies.

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Enhanced identification and biological validation of differential gene expression via Illumina whole-genome expression arrays through the use of the model-based background correction methodology.

Publication Date: 2008 May 1 PMID: 18450815
Authors: Ding, L. H. - Xie, Y. - Park, S. - Xiao, G. - Story, M. D.
Journal: Nucleic Acids Res

Despite the tremendous growth of microarray usage in scientific studies, there is a lack of standards for background correction methodologies, especially in single-color microarray platforms. Traditional background subtraction methods often generate negative signals and thus cause large amounts of data loss. Hence, some researchers prefer to avoid background corrections, which typically result in the underestimation of differential expression. Here, by utilizing nonspecific negative control features integrated into Illumina whole genome expression arrays, we have developed a method of model-based background correction for BeadArrays (MBCB). We compared the MBCB with a method adapted from the Affymetrix robust multi-array analysis algorithm and with no background subtraction, using a mouse acute myeloid leukemia (AML) dataset. We demonstrated that differential expression ratios obtained by using the MBCB had the best correlation with quantitative RT-PCR. MBCB also achieved better sensitivity in detecting differentially expressed genes with biological significance. For example, we demonstrated that the differential regulation of Tnfr2, Ikk and NF-kappaB, the death receptor pathway, in the AML samples, could only be detected by using data after MBCB implementation. We conclude that MBCB is a robust background correction method that will lead to more precise determination of gene expression and better biological interpretation of Illumina BeadArray data.

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Microarray-based global mapping of integration sites for the retrotransposon, intracisternal A-particle, in the mouse genome.

Publication Date: 2008 May 1 PMID: 18450814
Authors: Takabatake, T. - Ishihara, H. - Ohmachi, Y. - Tanaka, I. - Nakamura, M. M. - Fujikawa, K. - Hirouchi, T. - Kakinuma, S. - Shimada, Y. - Oghiso, Y. - Tanaka, K.
Journal: Nucleic Acids Res

Mammalian genomes contain numerous evolutionary harbored mobile elements, a part of which are still active and may cause genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression or disruption of neighboring host genes. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposon in a mammalian genome can be identified. Using this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA-integrated genomic regions, in which a considerable number of strain-specific putative insertions were included. In addition, by comparing genomic DNAs from radiation-induced myeloid leukemia cells and its reference normal tissue, we detected three genomic regions around which an IAP element was integrated. These results demonstrate the first successful genome-wide mapping of a retrotransposon type in a mammalian genome.

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Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons.

Publication Date: 2008 Apr 29 PMID: 18448472
Authors: Gundry, C. N. - Dobrowolski, S. F. - Martin, Y. R. - Robbins, T. C. - Nay, L. M. - Boyd, N. - Coyne, T. - Wall, M. D. - Wittwer, C. T. - Teng, D. H.
Journal: Nucleic Acids Res

Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise approximately 16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42-86 bp) genotyping on the LightScanner(R). Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average T(m) standard deviations of these targets decreased from 0.067 degrees C before calibration to 0.022 degrees C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086 degrees C to 0.032 degrees C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by T(m) even though current nearest neighbor models predict that the two homozygous alleles would be identical.

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Enzymatic synthesis of structure-free DNA with pseudo-complementary properties.

Publication Date: 2008 Apr 29 PMID: 18448471
Authors: Lahoud, G. - Timoshchuk, V. - Lebedev, A. - Vega, M. D. - Salas, M. - Arar, K. - Hou, Y. M. - Gamper, H.
Journal: Nucleic Acids Res

Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free single-stranded copies of any nucleic acid without impairing Watson-Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N(4)-alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that are <25 nt in length.

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Snap-to-it probes: chelate-constrained nucleobase oligomers with enhanced binding specificity.

Publication Date: 2008 Apr 29 PMID: 18448470
Authors: Morgan, J. R. - Lyon, R. P. - Maeda, D. Y. - Zebala, J. A.
Journal: Nucleic Acids Res

We describe snap-to-it probes, a novel probe technology to enhance the hybridization specificity of natural and unnatural nucleic acid oligomers using a simple and readily introduced structural motif. Snap-to-it probes were prepared from peptide nucleic acid (PNA) oligomers by modifying each terminus with a coordinating ligand. The two coordinating ligands constrain the probe into a macrocyclic configuration through formation of an intramolecular chelate with a divalent transition metal ion. On hybridization with a DNA target, the intramolecular chelate in the snap-to-it probe dissociates, resulting in the probe 'snapping-to' and binding the target nucleic acid. Thermal transition analysis of snap-to-it probes with complementary and single-mismatch DNA targets revealed that the transition between free and target-bound probe conformations was a reversible equilibrium, and the intramolecular chelate provided a thermodynamic barrier to target binding that resulted in a significant increase in mismatch discrimination. A 4-6 degrees C increase in specificity (DeltaT(m)) was observed from snap-to-it probes bearing either terminal iminodiacetic acid ligands coordinated with Ni(2+), or terminal dihistidine and nitrilotriacetic acid ligands coordinated with Cu(2+). The difference in specificity of the PNA oligomer relative to DNA was more than doubled in snap-to-it probes. Snap-to-it probes labeled with a fluorophore-quencher pair exhibited target-dependent fluorescence enhancement upon binding with target DNA.

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NOBAI: a web server for character coding of geometrical and statistical features in RNA structure.

Publication Date: 2008 Apr 29 PMID: 18448469
Authors: Knudsen, V. - Caetano-Anolles, G.
Journal: Nucleic Acids Res

The Numeration of Objects in Biology: Alignment Inferences (NOBAI) web server provides a web interface to the applications in the NOBAI software package. This software codes topological and thermodynamic information related to the secondary structure of RNA molecules as multi-state phylogenetic characters, builds character matrices directly in NEXUS format and provides sequence randomization options. The web server is an effective tool that facilitates the search for evolutionary history embedded in the structure of functional RNA molecules. The NOBAI web server is accessible at 'http://www.manet.uiuc.edu/nobai/nobai.php'. This web site is free and open to all users and there is no login requirement.

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MolAxis: a server for identification of channels in macromolecules.

Publication Date: 2008 Apr 29 PMID: 18448468
Authors: Yaffe, E. - Fishelovitch, D. - Wolfson, H. J. - Halperin, D. - Nussinov, R.
Journal: Nucleic Acids Res

MolAxis is a freely available, easy-to-use web server for identification of channels that connect buried cavities to the outside of macromolecules and for transmembrane (TM) channels in proteins. Biological channels are essential for physiological processes such as electrolyte and metabolite transport across membranes and enzyme catalysis, and can play a role in substrate specificity. Motivated by the importance of channel identification in macromolecules, we developed the MolAxis server. MolAxis implements state-of-the-art, accurate computational-geometry techniques that reduce the dimensions of the channel finding problem, rendering the algorithm extremely efficient. Given a protein or nucleic acid structure in the PDB format, the server outputs all possible channels that connect buried cavities to the outside of the protein or points to the main channel in TM proteins. For each channel, the gating residues and the narrowest radius termed 'bottleneck' are also given along with a full list of the lining residues and the channel surface in a 3D graphical representation. The users can manipulate advanced parameters and direct the channel search according to their needs. MolAxis is available as a web server or as a stand-alone program at http://bioinfo3d.cs.tau.ac.il/MolAxis.

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MassNet: a functional annotation service for protein mass spectrometry data.

Publication Date: 2008 Apr 29 PMID: 18448467
Authors: Park, D. - Kim, B. C. - Cho, S. W. - Park, S. J. - Choi, J. S. - Kim, S. I. - Bhak, J. - Lee, S.
Journal: Nucleic Acids Res

Although mass spectrometry has been frequently used to identify proteins, there are no web servers that provide comprehensive functional annotation of those identified proteins. It is necessary to provide such web service due to a rapid increase in the data. We, therefore, introduce MassNet, which provides (i) physico-chemical analysis information, (ii) KEGG pathway assignment (iii) Gene Ontology mapping and (iv) protein-protein interaction (PPI) prediction for the data from MASCOT, Prospector and Profound. MassNet provides the prediction information for PPIs using both 3D structural interaction and experimental interaction deposited in PSIMAP, BIND, DIP, HPRD, IntAct, MINT, CYGD and BioGrid. The web service is freely available at http://massnet.kr or http://sequenceome.kobic.re.kr/MassNet/.

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Identification of transcriptional regulatory cascades in retinoic acid-induced growth arrest of HepG2 cells.

Publication Date: 2008 Apr 29 PMID: 18445634
Authors: Nakanishi, M. - Tomaru, Y. - Miura, H. - Hayashizaki, Y. - Suzuki, M.
Journal: Nucleic Acids Res

All-trans retinoic acid (ATRA) is a potent inducer of cell differentiation and growth arrest. Here, we investigated ATRA-induced regulatory cascades associated with growth arrest of the human hepatoma cell line HepG2. ATRA induced >2-fold changes in the expression of 402 genes including 55 linked to cell-cycle regulation, cell growth or apoptosis during 48 h treatment. Computational search predicted that 250 transcriptional regulatory factors (TRFs) could recognize the proximal upstream regions of any of the 55 genes. Expression of 61 TRF genes was significantly changed during ATRA incubation, providing many potential regulatory edges. We focused on six TRFs that could regulate many of the 55 genes and found a total of 160 potential edges in which the expression of each of the genes was changed later than the expression change of the corresponding regulator. RNAi knockdown of the selected TRFs caused perturbation of the respective potential targets. The genes showed an opposite regulation pattern by ATRA and specific siRNA treatments were selected as strong candidates for direct TRF targets. Finally, 36 transcriptional regulatory edges were validated by chromatin immunoprecipitation. These analyses enabled us to depict a part of the transcriptional regulatory cascades closely linked to ATRA-induced cell growth arrest.

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A new pheromone trail-based genetic algorithm for comparative genome assembly.

Publication Date: 2008 Apr 29 PMID: 18445633
Authors: Zhao, F. - Zhao, F. - Li, T. - Bryant, D. A.
Journal: Nucleic Acids Res

Gap closing is considered one of the most challenging and time-consuming tasks in bacterial genome sequencing projects, especially with the emergence of new sequencing technologies, such as pyrosequencing, which may result in large amounts of data without the benefit of large insert libraries for contig scaffolding. We propose a novel algorithm to align contigs with more than one reference genome at a time. This approach can successfully overcome the limitations of low degrees of conserved gene order for the reference and target genomes. A pheromone trail-based genetic algorithm (PGA) was used to search globally for the optimal placement for each contig. Extensive testing on simulated and real data sets shows that PGA significantly outperforms previous methods, especially when assembling genomes that are only moderately related. An extended version of PGA can predict additional candidate connections for each contig and can thus increase the likelihood of identifying the correct arrangement of each contig. The software and test data sets can be accessed at http://sourceforge.net/projects/pga4genomics/.

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High-throughput functional annotation and data mining with the Blast2GO suite.

Publication Date: 2008 Apr 29 PMID: 18445632
Authors: Gotz, S. - Garcia-Gomez, J. M. - Terol, J. - Williams, T. D. - Nagaraj, S. H. - Nueda, M. J. - Robles, M. - Talon, M. - Dopazo, J. - Conesa, A.
Journal: Nucleic Acids Res

Functional genomics technologies have been widely adopted in the biological research of both model and non-model species. An efficient functional annotation of DNA or protein sequences is a major requirement for the successful application of these approaches as functional information on gene products is often the key to the interpretation of experimental results. Therefore, there is an increasing need for bioinformatics resources which are able to cope with large amount of sequence data, produce valuable annotation results and are easily accessible to laboratories where functional genomics projects are being undertaken. We present the Blast2GO suite as an integrated and biologist-oriented solution for the high-throughput and automatic functional annotation of DNA or protein sequences based on the Gene Ontology vocabulary. The most outstanding Blast2GO features are: (i) the combination of various annotation strategies and tools controlling type and intensity of annotation, (ii) the numerous graphical features such as the interactive GO-graph visualization for gene-set function profiling or descriptive charts, (iii) the general sequence management features and (iv) high-throughput capabilities. We used the Blast2GO framework to carry out a detailed analysis of annotation behaviour through homology transfer and its impact in functional genomics research. Our aim is to offer biologists useful information to take into account when addressing the task of functionally characterizing their sequence data.

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Insights into anti-termination regulation of the hut operon in Bacillus subtilis: importance of the dual RNA-binding surfaces of HutP.

Publication Date: 2008 Apr 29 PMID: 18445631
Authors: Gopinath, S. C. - Balasundaresan, D. - Kumarevel, T. - Misono, T. S. - Mizuno, H. - Kumar, P. K.
Journal: Nucleic Acids Res

The anti-termination protein, HutP, regulates the gene expression of the hut (histidine utilization) operon of Bacillus subtilis, by destabilizing the hut terminator RNA located upstream of the coding region encoding l-histidine degradation enzymes. On the basis of biochemical, in vivo and X-ray structural analyses, we now report that HutP uses its dual RNA-binding surfaces to access two XAG-rich regions (sites I and II) within the terminator RNA to mediate the destabilization process. In this process, HutP initiates destabilization at the 5'-end of its mRNA by binding to the first XAG-rich region (site I) and then accesses the second XAG-rich region (site II), located downstream of the stable G-C-rich segment of the terminator stem. By this action, HutP appears to disrupt the G-C-rich terminator stem, and thus prevents premature termination of transcription in the RNA segment preceding the regions encoding for the histidine degradation enzymes.

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Designating eukaryotic orthology via processed transcription units.

Publication Date: 2008 Apr 29 PMID: 18445630
Authors: Ho, M. R. - Jang, W. J. - Chen, C. H. - Ch'ang, L. Y. - Lin, W. C.
Journal: Nucleic Acids Res

Orthology is a widely used concept in comparative and evolutionary genomics. In addition to prokaryotic orthology, delineating eukaryotic orthology has provided insight into the evolution of higher organisms. Indeed, many eukaryotic ortholog databases have been established for this purpose. However, unlike prokaryotes, alternative splicing (AS) has hampered eukaryotic orthology assignments. Therefore, existing databases likely contain ambiguous eukaryotic ortholog relationships and possibly misclassify alternatively spliced protein isoforms as in-paralogs, which are duplicated genes that arise following speciation. Here, we propose a new approach for designating eukaryotic orthology using processed transcription units, and we present an orthology database prototype using the human and mouse genomes. Currently existing programs cover less than 69% of the human reference sequences when assigning human/mouse orthologs. In contrast, our method encompasses up to 80% of the human reference sequences. Moreover, the ortholog database presented herein is more than 92% consistent with the existing databases. In addition to managing AS, this approach is capable of identifying orthologs of embedded genes and fusion genes using syntenic evidence. In summary, this new approach is sensitive, specific and can generate a more comprehensive and accurate compilation of eukaryotic orthologs.

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Crystal structure of the 25 kDa subunit of human cleavage factor Im.

Publication Date: 2008 Apr 29 PMID: 18445629
Authors: Coseno, M. - Martin, G. - Berger, C. - Gilmartin, G. - Keller, W. - Doublie, S.
Journal: Nucleic Acids Res

Cleavage factor I(m) is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor I(m) is an oligomer composed of a small 25 kDa subunit (CF I(m)25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF I(m)25, a hydrolase motif with a characteristic alpha/beta/alpha fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF I(m)25 in its free and diadenosine tetraphosphate (Ap(4)A) bound forms at 1.85 and 1.80 A, respectively. CF I(m)25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF I(m)25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap(4)A. The complex and apo protein structures provide insight into the active oligomeric state of CF I(m) and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.

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A role for Caf1 in mRNA deadenylation and decay in trypanosomes and human cells.

Publication Date: 2008 Apr 27 PMID: 18442996
Authors: Schwede, A. - Ellis, L. - Luther, J. - Carrington, M. - Stoecklin, G. - Clayton, C.
Journal: Nucleic Acids Res

The eukaryotic Ccr4/Caf1/Not complex is involved in deadenylation of mRNAs. The Caf1 and Ccr4 subunits both potentially have deadenylating enzyme activity. We investigate here the roles of Ccr4 and Caf1 in deadenylation in two organisms that separated early in eukaryotic evolution: humans and trypanosomes. In Trypanosoma brucei, we found a complex containing CAF1, NOT1, NOT2 and NOT5, DHH1 and a possible homologue of Caf130; no homologue of Ccr4 was found. Trypanosome CAF1 has deadenylation activity, and is essential for cell survival. Depletion of trypanosome CAF1 delayed deadenylation and degradation of constitutively expressed mRNAs. Human cells have two isozymes of Caf1: simultaneous depletion of both inhibited degradation of an unstable reporter mRNA. In both species, depletion of Caf1 homologues inhibited deadenylation of bulk RNA and resulted in an increase in average poly(A) tail length.

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PVS: a web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery.

Publication Date: 2008 Apr 27 PMID: 18442995
Authors: Garcia-Boronat, M. - Diez-Rivero, C. M. - Reinherz, E. L. - Reche, P. A.
Journal: Nucleic Acids Res

We have developed PVS (Protein Variability Server), a web-based tool that uses several variability metrics to compute the absolute site variability in multiple protein-sequence alignments (MSAs). The variability is then assigned to a user-selected reference sequence consisting of either the first sequence in the alignment or a consensus sequence. Subsequently, PVS performs tasks that are relevant for structure-function studies, such as plotting and visualizing the variability in a relevant 3D-structure. Neatly, PVS also implements some other tasks that are thought to facilitate the design of epitope discovery-driven vaccines against pathogens where sequence variability largely contributes to immune evasion. Thus, PVS can return the conserved fragments in the MSA-as defined by a user-provided variability threshold-and locate them in a relevant 3D-structure. Furthermore, PVS can return a variability-masked sequence, which can be directly submitted to the RANKPEP server for the prediction of conserved T-cell epitopes. PVS is freely available at: http://imed.med.ucm.es/PVS/.

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