Nucleic Acids Research - Advance Access

Verification of specific G-quadruplex structure by using a novel cyanine dye supramolecular assembly: II. The binding characterization with specific intramolecular G-quadruplex and the recognizing mechanism

Thu, 19 Nov 2009 21:21:17 PST

The supramolecular assembly of a novel cyanine dye, 3,3'-di(3-sulfopropyl)-4,5,4',5'-dibenzo-9-ethyl-thiacarbocyanine triethylammonium salt (ETC) was designed to verify specific intramolecular G-quadruplexes from duplex and single-strand DNAs. Spectral results have shown that ETC presented two major distinct signatures with specific intramolecular G-quadruplexes in vitro: (i) dramatic changes in the absorption spectra (including disappearance of absorption peak around 660 nm and appearance of independent new peak around 584 nm); (ii) ~70 times enhancement of fluorescence signal at 600 nm. Furthermore, based on ¹H-nuclear magnetic resonance and circular dichroism results, the preferring binding of ETC to specific intramolecular G-quadruplexes probably result from end-stacking, and the loop structure nearby also plays an important role.

Adenovirus VA RNA-derived miRNAs target cellular genes involved in cell growth, gene expression and DNA repair

Thu, 19 Nov 2009 21:21:13 PST

Adenovirus virus-associated (VA) RNAs are processed to functional viral miRNAs or mivaRNAs. mivaRNAs are important for virus production, suggesting that they may target cellular or viral genes that affect the virus cell cycle. To look for cellular targets of mivaRNAs, we first identified genes downregulated in the presence of VA RNAs by microarray analysis. These genes were then screened for mivaRNA target sites using several bioinformatic tools. The combination of microarray analysis and bioinformatics allowed us to select the splicing and translation regulator TIA-1 as a putative mivaRNA target. We show that TIA-1 is downregulated at mRNA and protein levels in infected cells expressing functional mivaRNAs and in transfected cells that express mivaRNAI-138, one of the most abundant adenoviral miRNAs. Also, reporter assays show that TIA-1 is downregulated directly by mivaRNAI-138. To determine whether mivaRNAs could target other cellular genes we analyzed 50 additional putative targets. Thirty of them were downregulated in infected or transfected cells expressing mivaRNAs. Some of these genes are important for cell growth, transcription, RNA metabolism and DNA repair. We believe that a mivaRNA-mediated fine tune of the expression of some of these genes could be important in adenovirus cell cycle.

Correlated changes between regulatory cis elements and condition-specific expression in paralogous gene families

Singh, L. N., Hannenhalli, S. — Thu, 19 Nov 2009 21:21:20 PST

Gene duplication is integral to evolution, providing novel opportunities for organisms to diversify in function. One fundamental pathway of functional diversification among initially redundant gene copies, or paralogs, is via alterations in their expression patterns. Although the mechanisms underlying expression divergence are not completely understood, transcription factor binding sites and nucleosome occupancy are known to play a significant role in the process. Previous attempts to detect genomic variations mediating expression divergence in orthologs have had limited success for two primary reasons. First, it is inherently challenging to compare expressions among orthologs due to variable trans-acting effects and second, previous studies have quantified expression divergence in terms of an overall similarity of expression profiles across multiple samples, thereby obscuring condition-specific expression changes. Moreover, the inherently inter-correlated expressions among homologs present statistical challenges, not adequately addressed in many previous studies. Using rigorous statistical tests, here we characterize the relationship between cis element divergence and condition-specific expression divergence among paralogous genes in Saccharomyces cerevisiae. In particular, among all combinations of gene family and TFs analyzed, we found a significant correlation between TF binding and the condition-specific expression patterns in over 20% of the cases. In addition, incorporating nucleosome occupancy reveals several additional correlations. For instance, our results suggest that GAL4 binding plays a major role in the expression divergence of the genes in the sugar transporter family. Our work presents a novel means of investigating the cis regulatory changes potentially mediating expression divergence in paralogous gene families under specific conditions.

GeMMA: functional subfamily classification within superfamilies of predicted protein structural domains

Lee, D. A., Rentzsch, R., Orengo, C. — Wed, 18 Nov 2009 07:51:12 PST

GeMMA (Genome Modelling and Model Annotation) is a new approach to automatic functional subfamily classification within families and superfamilies of protein sequences. A major advantage of GeMMA is its ability to subclassify very large and diverse superfamilies with tens of thousands of members, without the need for an initial multiple sequence alignment. Its performance is shown to be comparable to the established high-performance method SCI-PHY. GeMMA follows an agglomerative clustering protocol that uses existing software for sensitive and accurate multiple sequence alignment and profile–profile comparison. The produced subfamilies are shown to be equivalent in quality whether whole protein sequences are used or just the sequences of component predicted structural domains. A faster, heuristic version of GeMMA that also uses distributed computing is shown to maintain the performance levels of the original implementation. The use of GeMMA to increase the functional annotation coverage of functionally diverse Pfam families is demonstrated. It is further shown how GeMMA clusters can help to predict the impact of experimentally determining a protein domain structure on comparative protein modelling coverage, in the context of structural genomics.

Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome

Albrecht, M., Sharma, C. M., Reinhardt, R., Vogel, J., Rudel, T. — Wed, 18 Nov 2009 07:51:07 PST

Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNA-seq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semi-quantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.

CAMP: a useful resource for research on antimicrobial peptides

Thomas, S., Karnik, S., Barai, R. S., Jayaraman, V. K., Idicula-Thomas, S. — Wed, 18 Nov 2009 07:51:04 PST

Antimicrobial peptides (AMPs) are gaining popularity as better substitute to antibiotics. These peptides are shown to be active against several bacteria, fungi, viruses, protozoa and cancerous cells. Understanding the role of primary structure of AMPs in their specificity and activity is essential for their rational design as drugs. Collection of Anti-Microbial Peptides (CAMP) is a free online database that has been developed for advancement of the present understanding on antimicrobial peptides. It is manually curated and currently holds 3782 antimicrobial sequences. These sequences are divided into experimentally validated (patents and non-patents: 2766) and predicted (1016) datasets based on their reference literature. Information like source organism, activity (MIC values), reference literature, target and non-target organisms of AMPs are captured in the database. The experimentally validated dataset has been further used to develop prediction tools for AMPs based on the machine learning algorithms like Random Forests (RF), Support Vector Machines (SVM) and Discriminant Analysis (DA). The prediction models gave accuracies of 93.2% (RF), 91.5% (SVM) and 87.5% (DA) on the test datasets. The prediction and sequence analysis tools, including BLAST, are integrated in the database. CAMP will be a useful database for study of sequence-activity and -specificity relationships in AMPs. CAMP is freely available at http://www.bicnirrh.res.in/antimicrobial.

Sequence-structure relationships in RNA loops: establishing the basis for loop homology modeling

Schudoma, C., May, P., Nikiforova, V., Walther, D. — Wed, 18 Nov 2009 07:51:00 PST

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence–structure relationships in loops. Loops differing by <25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

FunSimMat update: new features for exploring functional similarity

Schlicker, A., Albrecht, M. — Wed, 18 Nov 2009 07:51:23 PST

Quantifying the functional similarity of genes and their products based on Gene Ontology annotation is an important tool for diverse applications like the analysis of gene expression data, the prediction and validation of protein functions and interactions, and the prioritization of disease genes. The Functional Similarity Matrix (FunSimMat, http://www.funsimmat.de) is a comprehensive database providing various precomputed functional similarity values for proteins in UniProtKB and for protein families in Pfam and SMART. With this update, we significantly increase the coverage of FunSimMat by adding data from the Gene Ontology Annotation project as well as new functional similarity measures. The applicability of the database is greatly extended by the implementation of a new Gene Ontology-based method for disease gene prioritization. Two new visualization tools allow an interactive analysis of the functional relationships between proteins or protein families. This is enhanced further by the introduction of an automatically derived hierarchy of annotation classes. Additional changes include a revised user front-end and a new RESTlike interface for improving the user-friendliness and online accessibility of FunSimMat.

A re-annotation pipeline for Illumina BeadArrays: improving the interpretation of gene expression data

Wed, 18 Nov 2009 07:51:20 PST

Illumina BeadArrays are among the most popular and reliable platforms for gene expression profiling. However, little external scrutiny has been given to the design, selection and annotation of BeadArray probes, which is a fundamental issue in data quality and interpretation. Here we present a pipeline for the complete genomic and transcriptomic re-annotation of Illumina probe sequences, also applicable to other platforms, with its output available through a Web interface and incorporated into Bioconductor packages. We have identified several problems with the design of individual probes and we show the benefits of probe re-annotation on the analysis of BeadArray gene expression data sets. We discuss the importance of aspects such as probe coverage of individual transcripts, alternative messenger RNA splicing, single-nucleotide polymorphisms, repeat sequences, RNA degradation biases and probes targeting genomic regions with no known transcription. We conclude that many of the Illumina probes have unreliable original annotation and that our re-annotation allows analyses to focus on the good quality probes, which form the majority, and also to expand the scope of biological information that can be extracted.

BioDrugScreen: a computational drug design resource for ranking molecules docked to the human proteome

Wed, 18 Nov 2009 07:51:18 PST

BioDrugScreen is a resource for ranking molecules docked against a large number of targets in the human proteome. Nearly 1600 molecules from the freely available NCI diversity set were docked onto 1926 cavities identified on 1589 human targets resulting in >3 million receptor–ligand complexes requiring >200 000 cpu-hours on the TeraGrid. The targets in BioDrugScreen originated from Human Cancer Protein Interaction Network, which we have updated, as well as the Human Druggable Proteome, which we have created for the purpose of this effort. This makes the BioDrugScreen resource highly valuable in drug discovery. The receptor–ligand complexes within the database can be ranked using standard and well-established scoring functions like AutoDock, DockScore, ChemScore, X-Score, GoldScore, DFIRE and PMF. In addition, we have scored the complexes with more intensive GBSA and PBSA approaches requiring an additional 120 000 cpu-hours on the TeraGrid. We constructed a simple interface to enable users to view top-ranking molecules and access purchasing and other information for further experimental exploration.

An Alu-derived intronic splicing enhancer facilitates intronic processing and modulates aberrant splicing in ATM

Pastor, T., Talotti, G., Lewandowska, M. A., Pagani, F. — Wed, 18 Nov 2009 07:51:15 PST

We have previously reported a natural GTAA deletion within an intronic splicing processing element (ISPE) of the ataxia telangiectasia mutated (ATM) gene that disrupts a non-canonical U1 snRNP interaction and activates the excision of the upstream portion of the intron. The resulting pre-mRNA splicing intermediate is then processed to a cryptic exon, whose aberrant inclusion in the final mRNA is responsible for ataxia telangiectasia. We show here that the last 40 bases of a downstream intronic antisense Alu repeat are required for the activation of the cryptic exon by the ISPE deletion. Evaluation of the pre-mRNA splicing intermediate by a hybrid minigene assay indicates that the identified intronic splicing enhancer represents a novel class of enhancers that facilitates processing of splicing intermediates possibly by recruiting U1 snRNP to defective donor sites. In the absence of this element, the splicing intermediate accumulates and is not further processed to generate the cryptic exon. Our results indicate that Alu-derived sequences can provide intronic splicing regulatory elements that facilitate pre-mRNA processing and potentially affect the severity of disease-causing splicing mutations.

A profusion of upstream open reading frame mechanisms in polyamine-responsive translational regulation

Ivanov, I. P., Atkins, J. F., Michael, A. J. — Tue, 17 Nov 2009 08:17:03 PST

In many eukaryotic mRNAs one or more short ‘upstream’ open reading frames, uORFs, precede the initiator of the main coding sequence. Upstream ORFs are functionally diverse as illustrated by their variety of features in polyamine pathway biosynthetic mRNAs. Their propensity to act as sensors for regulatory circuits and to amplify the signals likely explains their occurrence in most polyamine pathway mRNAs. The uORF-mediated polyamine responsive autoregulatory circuits found in polyamine pathway mRNAs exemplify the translationally regulated dynamic interface between components of the proteome and metabolism.

A single-molecule assay for telomerase structure-function analysis

Wu, J. Y., Stone, M. D., Zhuang, X. — Tue, 17 Nov 2009 08:17:00 PST

The activity of the telomerase ribonucleoprotein enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures.

The Negatome database: a reference set of non-interacting protein pairs

Tue, 17 Nov 2009 08:16:58 PST

The Negatome is a collection of protein and domain pairs that are unlikely to be engaged in direct physical interactions. The database currently contains experimentally supported non-interacting protein pairs derived from two distinct sources: by manual curation of literature and by analyzing protein complexes with known 3D structure. More stringent lists of non-interacting pairs were derived from these two datasets by excluding interactions detected by high-throughput approaches. Additionally, non-interacting protein domains have been derived from the stringent manual and structural data, respectively. The Negatome is much less biased toward functionally dissimilar proteins than the negative data derived by randomly selecting proteins from different cellular locations. It can be used to evaluate protein and domain interactions from new experiments and improve the training of interaction prediction algorithms. The Negatome database is available at http://mips.helmholtz-muenchen.de/proj/ppi/negatome.

The Gene Ontology in 2010: extensions and refinements

The Gene Ontology Consortium — Tue, 17 Nov 2009 08:16:55 PST

The Gene Ontology (GO) Consortium (http://www.geneontology.org) (GOC) continues to develop, maintain and use a set of structured, controlled vocabularies for the annotation of genes, gene products and sequences. The GO ontologies are expanding both in content and in structure. Several new relationship types have been introduced and used, along with existing relationships, to create links between and within the GO domains. These improve the representation of biology, facilitate querying, and allow GO developers to systematically check for and correct inconsistencies within the GO. Gene product annotation using GO continues to increase both in the number of total annotations and in species coverage. GO tools, such as OBO-Edit, an ontology-editing tool, and AmiGO, the GOC ontology browser, have seen major improvements in functionality, speed and ease of use.

ELM: the status of the 2010 eukaryotic linear motif resource

Tue, 17 Nov 2009 08:16:53 PST

Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a ‘Bar Code’ format, which also displays known instances from homologous proteins through a novel ‘Instance Mapper’ protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation.

Extensive mis-splicing of a bi-partite plant mitochondrial group II intron

Elina, H., Brown, G. G. — Tue, 17 Nov 2009 08:17:25 PST

Expression of the seed plant mitochondrial nad5 gene involves two trans-splicing events that remove fragmented group II introns and join the small, central exon c to exons b and d. We show that in both monocot and eudicot plants, extensive mis-splicing of the bi-partite intron 2 takes place, resulting in the formation of aberrantly spliced products in which exon c is joined to various sites within exon b. These mis-spliced products accumulate to levels comparable to or greater than that of the correctly spliced mRNA. We suggest that mis-splicing may result from folding constraints imposed on intron 2 by base-pairing between exon a and a portion of the bi-partite intron 3 downstream of exon c. Consistent with this hypothesis, we find that mis-splicing does not occur in Oenothera mitochondria, where intron 3 is further fragmented such that the predicted base-pairing region is not covalently linked to exon c. Our findings suggest that intron fragmentation may lead to mis-splicing, which may be corrected by further intron fragmentation.

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

Ghosh, S. K., Huang, C.-C., Hajra, S., Jayaram, M. — Tue, 17 Nov 2009 08:17:23 PST

Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin’s embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres.

The Pfam protein families database

Tue, 17 Nov 2009 08:17:20 PST

Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version of the popular profile hidden Markov model package. This software is ~100 times faster than HMMER2 and is more sensitive due to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described in detail. Pfam release 24.0 contains 11 912 families, of which a large number have been significantly updated during the past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/).

The ITS2 Database III--sequences and structures for phylogeny

Tue, 17 Nov 2009 08:17:17 PST

The internal transcribed spacer 2 (ITS2) is a widely used phylogenetic marker. In the past, it has mainly been used for species level classifications. Nowadays, a wider applicability becomes apparent. Here, the conserved structure of the RNA molecule plays a vital role. We have developed the ITS2 Database (http://its2.bioapps.biozentrum.uni-wuerzburg.de) which holds information about sequence, structure and taxonomic classification of all ITS2 in GenBank. In the new version, we use Hidden Markov models (HMMs) for the identification and delineation of the ITS2 resulting in a major redesign of the annotation pipeline. This allowed the identification of more than 160 000 correct full length and more than 50 000 partial structures. In the web interface, these can now be searched with a modified BLAST considering both sequence and structure, enabling rapid taxon sampling. Novel sequences can be annotated using the HMM based approach and modelled according to multiple template structures. Sequences can be searched for known and newly identified motifs. Together, the database and the web server build an exhaustive resource for ITS2 based phylogenetic analyses.

Intrinsic flexibility of B-DNA: the experimental TRX scale

Heddi, B., Oguey, C., Lavelle, C., Foloppe, N., Hartmann, B. — Tue, 17 Nov 2009 08:17:14 PST

B-DNA flexibility, crucial for DNA–protein recognition, is sequence dependent. Free DNA in solution would in principle be the best reference state to uncover the relation between base sequences and their intrinsic flexibility; however, this has long been hampered by a lack of suitable experimental data. We investigated this relationship by compiling and analyzing a large dataset of NMR ³¹P chemical shifts in solution. These measurements reflect the BI BII equilibrium in DNA, intimately correlated to helicoidal descriptors of the curvature, winding and groove dimensions. Comparing the ten complementary DNA dinucleotide steps indicates that some steps are much more flexible than others. This malleability is primarily controlled at the dinucleotide level, modulated by the tetranucleotide environment. Our analyses provide an experimental scale called TRX that quantifies the intrinsic flexibility of the ten dinucleotide steps in terms of Twist, Roll, and X-disp (base pair displacement). Applying the TRX scale to DNA sequences optimized for nucleosome formation reveals a 10 base-pair periodic alternation of stiff and flexible regions. Thus, DNA flexibility captured by the TRX scale is relevant to nucleosome formation, suggesting that this scale may be of general interest to better understand protein-DNA recognition.

ENCODE whole-genome data in the UCSC Genome Browser

Tue, 17 Nov 2009 08:17:11 PST

The Encyclopedia of DNA Elements (ENCODE) project is an international consortium of investigators funded to analyze the human genome with the goal of producing a comprehensive catalog of functional elements. The ENCODE Data Coordination Center at The University of California, Santa Cruz (UCSC) is the primary repository for experimental results generated by ENCODE investigators. These results are captured in the UCSC Genome Bioinformatics database and download server for visualization and data mining via the UCSC Genome Browser and companion tools (Rhead et al. The UCSC Genome Browser Database: update 2010, in this issue). The ENCODE web portal at UCSC (http://encodeproject.org or http://genome.ucsc.edu/ENCODE) provides information about the ENCODE data and convenient links for access.

IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF

Ehrenmann, F., Kaas, Q., Lefranc, M.-P. — Tue, 17 Nov 2009 08:17:09 PST

IMGT/3Dstructure-DB is the three-dimensional (3D) structure database of IMGT^®, the international ImMunoGenetics information system^® that is acknowledged as the global reference in immunogenetics and immunoinformatics. IMGT/3Dstructure-DB contains 3D structures of immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility complex (MHC) proteins, antigen receptor/antigen complexes (IG/Ag, TR/peptide/MHC) of vertebrates; 3D structures of related proteins of the immune system (RPI) of vertebrates and invertebrates, belonging to the immunoglobulin and MHC superfamilies (IgSF and MhcSF, respectively) and found in complexes with IG, TR or MHC. IMGT/3Dstructure-DB data are annotated according to the IMGT criteria, using IMGT/DomainGapAlign, and based on the IMGT-ONTOLOGY concepts and axioms. IMGT/3Dstructure-DB provides IMGT gene and allele identification (CLASSIFICATION), region and domain delimitations (DESCRIPTION), amino acid positions according to the IMGT unique numbering (NUMEROTATION) that are used in IMGT/3Dstructure-DB cards, results of contact analysis and renumbered flat files. In its Web version, the IMGT/DomainGapAlign tool analyses amino acid sequences, per domain. Coupled to the IMGT/Collier-de-Perles tool, it provides an invaluable help for antibody engineering and humanization design based on complementarity determining region (CDR) grafting as it precisely defines the standardized framework regions (FR-IMGT) and CDR-IMGT. IMGT/3Dstructure-DB and IMGT/DomainGapAlign are freely available at http://www.imgt.org.

miRGen 2.0: a database of microRNA genomic information and regulation

Tue, 17 Nov 2009 08:17:06 PST

MicroRNAs are small, non-protein coding RNA molecules known to regulate the expression of genes by binding to the 3'UTR region of mRNAs. MicroRNAs are produced from longer transcripts which can code for more than one mature miRNAs. miRGen 2.0 is a database that aims to provide comprehensive information about the position of human and mouse microRNA coding transcripts and their regulation by transcription factors, including a unique compilation of both predicted and experimentally supported data. Expression profiles of microRNAs in several tissues and cell lines, single nucleotide polymorphism locations, microRNA target prediction on protein coding genes and mapping of miRNA targets of co-regulated miRNAs on biological pathways are also integrated into the database and user interface. The miRGen database will be continuously maintained and freely available at http://www.microrna.gr/mirgen/.

IRESite--a tool for the examination of viral and cellular internal ribosome entry sites

Mokrejs, M., Masek, T., Vopalensky, V., Hlubucek, P., Delbos, P., Pospisek, M. — Mon, 16 Nov 2009 07:16:24 PST

The IRESite (http://www.iresite.org) presents carefully curated experimental evidence of many eukaryotic viral and cellular internal ribosome entry site (IRES) regions. At the time of submission, IRESite stored >600 records. The IRESite gradually evolved into a robust tool providing (i) biologically meaningful information regarding the IRESs and their experimental background (including annotation of IRES secondary structures and IRES trans-acting factors) as well as (ii) thorough concluding remarks to stored database entries and regularly updated evaluation of the reported IRES function. A substantial portion of the IRESite data results purely from in-house bioinformatic analyses of currently available sequences, in silico attempts to repeat published cloning experiments, DNA sequencing and restriction endonuclease verification of received plasmid DNA. We also present a newly implemented tool for displaying RNA secondary structures and for searching through the structures currently stored in the database. The supplementary material contains an updated list of reported IRESs.

Analysis of acyclic nucleoside modifications in siRNAs finds sensitivity at position 1 that is restored by 5'-terminal phosphorylation both in vitro and in vivo

Mon, 16 Nov 2009 07:16:21 PST

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that use the endogenous RNAi pathway to mediate gene silencing. Phosphorylation facilitates loading of a siRNA into the Ago2 complex and subsequent cleavage of the target mRNA. In this study, 2', 3' seco nucleoside modifications, which contain an acylic ribose ring and are commonly called unlocked nucleic acids (UNAs), were evaluated at all positions along the guide strand of a siRNA targeting apolipoprotein B (ApoB). UNA modifications at positions 1, 2 and 3 were detrimental to siRNA activity. UNAs at positions 1 and 2 prevented phosphorylation by Clp1 kinase, abrogated binding to Ago2, and impaired Ago2-mediated cleavage of the mRNA target. The addition of a 5'-terminal phosphate to siRNA containing a position 1 UNA restored ApoB mRNA silencing, Ago2 binding, and Ago2 mediated cleavage activity. Position 1 UNA modified siRNA containing a 5'-terminal phosphate exhibited a partial restoration of siRNA silencing activity in vivo. These data reveal the complexity of interpreting the effects of chemical modification on siRNA activity, and exemplify the importance of using multiple biochemical, cell-based and in vivo assays to rationally design chemically modified siRNA destined for therapeutic use.

Probing the (H3-H4)2 histone tetramer structure using pulsed EPR spectroscopy combined with site-directed spin labelling

Bowman, A., Ward, R., El-Mkami, H., Owen-Hughes, T., Norman, D. G. — Fri, 13 Nov 2009 03:32:08 PST

The (H3-H4)₂ histone tetramer forms the central core of nucleosomes and, as such, plays a prominent role in assembly, disassembly and positioning of nucleosomes. Despite its fundamental role in chromatin, the tetramer has received little structural investigation. Here, through the use of pulsed electron-electron double resonance spectroscopy coupled with site-directed spin labelling, we survey the structure of the tetramer in solution. We find that tetramer is structurally more heterogeneous on its own than when sequestered in the octamer or nucleosome. In particular, while the central region including the H3-H3' interface retains a structure similar to that observed in nucleosomes, other regions such as the H3 N helix display increased structural heterogeneity. Flexibility of the H3 N helix in the free tetramer also illustrates the potential for post-translational modifications to alter the structure of this region and mediate interactions with histone chaperones. The approach described here promises to prove a powerful system for investigating the structure of additional assemblies of histones with other important factors in chromatin assembly/fluidity.

The relaxed requirements of the integron cleavage site allow predictable changes in integron target specificity

Frumerie, C., Ducos-Galand, M., Gopaul, D. N., Mazel, D. — Fri, 13 Nov 2009 03:32:17 PST

Integrons are able to incorporate exogenous genes embedded in mobile cassettes, by a site-specific recombination mechanism. Gene cassettes are collected at the attI site, via an integrase mediated recombination between the cassette recombination site, attC, and the attI site. Interestingly, only three nucleotides are conserved between attC and attI. Here, we have determined the requirements of these in recombination, using the recombination machinery from the paradigmatic class 1 integron. We found that, strikingly, the only requirement is to have identical first nucleotide in the two partner sites, but not the nature of this nucleotide. Furthermore, we showed that the reaction is close to wild-type efficiency when one of the nucleotides in the second or third position is mutated in either the attC or the attI1 site, while identical mutations can have drastic effects when both sites are mutated, resulting in a dramatic decrease of recombination frequency compared to that of the wild-type sites. Finally, we tested the functional role of the amino acids predicted from structural data to interact with the cleavage site. We found that, if the recombination site triplets are tolerant to mutation, the amino acids interacting with them are extremely constrained.

EuPathDB: a portal to eukaryotic pathogen databases

Fri, 13 Nov 2009 03:32:14 PST

EuPathDB (http://EuPathDB.org; formerly ApiDB) is an integrated database covering the eukaryotic pathogens of the genera Cryptosporidium, Giardia, Leishmania, Neospora, Plasmodium, Toxoplasma, Trichomonas and Trypanosoma. While each of these groups is supported by a taxon-specific database built upon the same infrastructure, the EuPathDB portal offers an entry point to all these resources, and the opportunity to leverage orthology for searches across genera. The most recent release of EuPathDB includes updates and changes affecting data content, infrastructure and the user interface, improving data access and enhancing the user experience. EuPathDB currently supports more than 80 searches and the recently-implemented ‘search strategy’ system enables users to construct complex multi-step searches via a graphical interface. Search results are dynamically displayed as the strategy is constructed or modified, and can be downloaded, saved, revised, or shared with other database users.

The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata

Fri, 13 Nov 2009 03:32:11 PST

The Genomes On Line Database (GOLD) is a comprehensive resource for centralized monitoring of genome and metagenome projects worldwide. Both complete and ongoing projects, along with their associated metadata, can be accessed in GOLD through precomputed tables and a search page. As of September 2009, GOLD contains information for more than 5800 sequencing projects, of which 1100 have been completed and their sequence data deposited in a public repository. GOLD continues to expand, moving toward the goal of providing the most comprehensive repository of metadata information related to the projects and their organisms/environments in accordance with the Minimum Information about a (Meta)Genome Sequence (MIGS/MIMS) specification. GOLD is available at: http://www.genomesonline.org and has a mirror site at the Institute of Molecular Biology and Biotechnology, Crete, Greece, at: http://gold.imbb.forth.gr/

MeMotif: a database of linear motifs in {alpha}-helical transmembrane proteins

Thu, 12 Nov 2009 01:29:58 PST

Membrane proteins are important for many processes in the cell and used as main drug targets. The increasing number of high-resolution structures available makes for the first time a characterization of local structural and functional motifs in -helical transmembrane proteins possible. MeMotif (http://projects.biotec.tu-dresden.de/memotif) is a database and wiki which collects more than 2000 known and novel computationally predicted linear motifs in -helical transmembrane proteins. Motifs are fully described in terms of several structural and functional features and editable. Motifs contained in MeMotif can be used in different biological applications, from the identification of biochemically important functional residues which are candidates for mutagenesis experiments to the improvement of tools for transmembrane protein modeling.

Functional properties and evolutionary splicing constraints on a composite exonic regulatory element of splicing in CFTR exon 12

Haque, A., Buratti, E., Baralle, F. E. — Thu, 12 Nov 2009 01:29:55 PST

In general, splicing regulatory elements are defined as Enhancers or Silencers depending on their positive or negative effect upon exon inclusion. Often, these sequences are usually present separate from each other in exonic/intronic sequences. The Composite Exonic Splicing Regulatory Elements (CERES) represent an extreme physical overlap of enhancer/silencer activity. As a result, when CERES elements are mutated the consequences on the splicing process are difficult to predict. Here, we show that the functional activity of the CERES2 sequence in CFTR exon 12 is regulated by the binding, in very close proximity to each other, of several SR and hnRNP proteins. Moreover, our results show that practically the entire exon 12 sequence context participate in its definition. The consequences of this situation can be observed at the evolutionary level by comparing changes in conservation of different splicing elements in different species. In conclusion, our study highlights how it is increasingly difficult to define many exonic sequences by simply breaking them down in isolated enhancer/silencer or even neutral elements. The real picture is close to one of continuous competition between positive and negative factors where affinity for the target sequences and other dynamic factors decide the inclusion or exclusion of the exon.

GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 update

Gilsdorf, M., Horn, T., Arziman, Z., Pelz, O., Kiner, E., Boutros, M. — Thu, 12 Nov 2009 06:41:59 PST

The GenomeRNAi database (http://www.genomernai.org/) contains phenotypes from published cell-based RNA interference (RNAi) screens in Drosophila and Homo sapiens. The database connects observed phenotypes with annotations of targeted genes and information about the RNAi reagent used for the perturbation experiment. The availability of phenotypes from Drosophila and human screens also allows for phenotype searches across species. Besides reporting quantitative data from genome-scale screens, the new release of GenomeRNAi also enables reporting of data from microscopy experiments and curated phenotypes from published screens. In addition, the database provides an updated resource of RNAi reagents and their predicted quality that are available for the Drosophila and the human genome. The new version also facilitates the integration with other genomic data sets and contains expression profiling (RNA-Seq) data for several cell lines commonly used in RNAi experiments.

Interaction of Borrelia burgdorferi Hbb with the p66 promoter

Medrano, M. S., Policastro, P. F., Schwan, T. G., Coburn, J. — Thu, 12 Nov 2009 01:29:52 PST

Borrelia burgdorferi, an agent of Lyme disease, encodes the β₃-chain integrin ligand P66. P66 is expressed by B. burgdorferi in the mammal, in laboratory media, and as the bacteria are acquired or transmitted by the tick, but is not expressed by the bacterium in unfed ticks. Attempts to reveal factors influencing expression revealed that P66 was expressed in all in vitro conditions investigated. Candidate regulators identified in a search of the B. burgdorferi genome for homologs to other bacterial transcription factors were cloned and introduced into E. coli carrying a p66 promoter-signal sequence-phoA (alkaline phosphatase, or AP) fusion. Three candidate transcription factors—two that decreased AP activity (Hbb and BB0527), and one that increased AP activity (BBA23)—were identified. BBA23 and BB0527 did not bind to the p66 promoter at physiologically relevant concentrations. In contrast, several promoter fragments, including p66, were bound by Hbb (BB0232), with slightly different affinities. Consistent with results from other laboratories, Hbb appears to recognize multiple DNA sequences. Changes in the expression of p66 and bb0232 in the tick at various points with respect to feeding on mice, along with the results of the reporter experiment in the surrogate host E. coli, are consistent with Hbb/BB0232 being involved in regulating p66 expression.

Quantitative analysis of ribosome-mRNA complexes at different translation stages

Thu, 12 Nov 2009 01:29:49 PST

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture.

GenBank

Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., Sayers, E. W. — Thu, 12 Nov 2009 06:41:54 PST

GenBank® is a comprehensive database that contains publicly available nucleotide sequences for more than 300 000 organisms named at the genus level or lower, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or standalone Sequin programs, and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the European Molecular Biology Laboratory Nucleotide Sequence Database in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through the NCBI Entrez retrieval system, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bi-monthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI homepage: www.ncbi.nlm.nih.gov.

Independent mechanisms of stimulation of polynucleotide kinase/phosphatase by phosphorylated and non-phosphorylated XRCC1

Thu, 12 Nov 2009 01:29:44 PST

XRCC1 plays a central role in mammalian single-strand break repair. Although it has no enzymatic activity of its own, it stimulates the activities of polynucleotide kinase/phosphatase (PNKP), and this function is enhanced by protein kinase CK2 mediated phosphorylation of XRCC1. We have previously shown that non-phosphorylated XRCC1 stimulates the kinase activity of PNKP by increasing the turnover of PNKP. Here we extend our analysis of the XRCC1-PNKP interaction taking into account the phosphorylation of XRCC1. We demonstrate that phosphorylated and non-phosphorylated XRCC1 interact with different regions of PNKP. Phosphorylated XRCC1 binds with high affinity (K_d = 3.5 nM and 1 : 1 stoichiometry) to the forkhead associated (FHA) domain, while non-phosphorylated XRCC1 binds to the catalytic domain of PNKP with lower affinity (K_d = 43.0 nM and 1 : 1 stoichiometry). Under conditions of limited enzyme concentration both forms of XRCC1 enhance the activities of PNKP, but the effect is more pronounced with phosphorylated XRCC1, particularly for the kinase activity of PNKP. The stimulatory effect of phosphorylated XRCC1 on PNKP can be totally inhibited by the presence of excess FHA domain polypeptide, but non-phosphorylated XRCC1 is not susceptible to competition by the FHA domain. Thus, XRCC1 can stimulate PNKP by two independent mechanisms.

DBTSS provides a tissue specific dynamic view of Transcription Start Sites

Yamashita, R., Wakaguri, H., Sugano, S., Suzuki, Y., Nakai, K. — Thu, 12 Nov 2009 01:29:40 PST

DataBase of Transcription Start Sites (DBTSS) is a database which contains precise positional information for transcription start sites (TSSs) of eukaryotic mRNAs. In this update, we included 330 million new tags generated by massively sequencing the 5'-end of oligo-cap selected cDNAs in humans and mice. The tags were collected from normal fetal or adult human tissues, including brain, thymus, liver, kidney and heart, from 6 human cell lines in 21 diverse growth conditions as well as from mouse NIH3T3 cell line: altogether 31 different cell types or culture conditions are represented. This unprecedented increase in depth of data now allows DBTSS to faithfully represent the dynamically changing landscape of TSSs in different cell types and conditions, during development and in the course of evolution. Differential usage of alternative 5'-ends across cell types and conditions can be viewed in a series of new interfaces. Promoter sequence information is now displayed in a comparative genomics viewer where evolutionary turnover of the TSSs can be evaluated. DBTSS can be accessed at http://dbtss.hgc.jp/.

HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation

Thu, 12 Nov 2009 01:29:37 PST

The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K_d = 27 ± 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

Database resources of the National Center for Biotechnology Information

Thu, 12 Nov 2009 06:42:07 PST

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, Reference Sequence, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Entrez Probe, GENSAT, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool, Biosystems, Peptidome, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. All these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

WormBase: a comprehensive resource for nematode research

Thu, 12 Nov 2009 06:42:04 PST

WormBase (http://www.wormbase.org) is a central data repository for nematode biology. Initially created as a service to the Caenorhabditis elegans research field, WormBase has evolved into a powerful research tool in its own right. In the past 2 years, we expanded WormBase to include the complete genomic sequence, gene predictions and orthology assignments from a range of related nematodes. This comparative data enrich the C. elegans data with improved gene predictions and a better understanding of gene function. In turn, they bring the wealth of experimental knowledge of C. elegans to other systems of medical and agricultural importance. Here, we describe new species and data types now available at WormBase. In addition, we detail enhancements to our curatorial pipeline and website infrastructure to accommodate new genomes and an extensive user base.

The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

Dong, J., George, N. P., Duckett, K. L., DeBeer, M. A. P., Lopper, M. E. — Wed, 11 Nov 2009 04:48:57 PST

Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 Å resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.

Divergent human remodeling complexes remove nucleosomes from strong positioning sequences

Pham, C. D., He, X., Schnitzler, G. R. — Wed, 11 Nov 2009 04:48:54 PST

Nucleosome positioning plays a major role in controlling the accessibility of DNA to transcription factors and other nuclear processes. Nucleosome positions after assembly are at least partially determined by the relative affinity of DNA sequences for the histone octamer. Nucleosomes can be moved, however, by a class of ATP dependent chromatin remodeling complexes. We recently showed that the human SWI/SNF remodeling complex moves nucleosomes in a sequence specific manner, away from nucleosome positioning sequences (NPSes). Here, we compare the repositioning specificity of five remodelers of diverse biological functions (hSWI/SNF, the SNF2h ATPase and the hACF, CHRAC and WICH complexes than each contain SNF2h) on 5S rDNA, MMTV and 601 NPS polynucleosomal templates. We find that all five remodelers act similarly to reduce nucleosome occupancy over the strongest NPSes, an effect that could directly contribute to the function of WICH in activating 5S rDNA transcription. While some differences were observed between complexes, all five remodelers were found to result in surprisingly similar nucleosome distributions. This suggests that remodeling complexes may share a conserved repositioning specificity, and that their divergent biological functions may largely arise from other properties conferred by complex-specific subunits.

Protein Geometry Database: a flexible engine to explore backbone conformations and their relationships to covalent geometry

Berkholz, D. S., Krenesky, P. B., Davidson, J. R., Karplus, P. A. — Wed, 11 Nov 2009 01:35:28 PST

The backbone bond lengths, bond angles, and planarity of a protein are influenced by the backbone conformation (,), but no tool exists to explore these relationships, leaving this area as a reservoir of untapped information about protein structure and function. The Protein Geometry Database (PGD) enables biologists to easily and flexibly query information about the conformation alone, the backbone geometry alone, and the relationships between them. The capabilities the PGD provides are valuable for assessing the uniqueness of observed conformational or geometric features in protein structure as well as discovering novel features and principles of protein structure. The PGD server is available at http://pgd.science.oregonstate.edu/ and the data and code underlying it are freely available to use and extend.

Sole-Search: an integrated analysis program for peak detection and functional annotation using ChIP-seq data

Wed, 11 Nov 2009 04:48:52 PST

Next-generation sequencing is revolutionizing the identification of transcription factor binding sites throughout the human genome. However, the bioinformatics analysis of large datasets collected using chromatin immunoprecipitation and high-throughput sequencing is often a roadblock that impedes researchers in their attempts to gain biological insights from their experiments. We have developed integrated peak-calling and analysis software (Sole-Search) which is available through a user-friendly interface and (i) converts raw data into a format for visualization on a genome browser, (ii) outputs ranked peak locations using a statistically based method that overcomes the significant problem of false positives, (iii) identifies the gene nearest to each peak, (iv) classifies the location of each peak relative to gene structure, (v) provides information such as the number of binding sites per chromosome and per gene and (vi) allows the user to determine overlap between two different experiments. In addition, the program performs an analysis of amplified and deleted regions of the input genome. This software is web-based and automated, allowing easy and immediate access to all investigators. We demonstrate the utility of our software by collecting, analyzing and comparing ChIP-seq data for six different human transcription factors/cell line combinations.

Sumoylation of the BLM ortholog, Sgs1, promotes telomere-telomere recombination in budding yeast

Lu, C.-Y., Tsai, C.-H., Brill, S. J., Teng, S.-C. — Wed, 11 Nov 2009 05:51:21 PST

BLM and WRN are members of the RecQ family of DNA helicases, and in humans their loss is associated with syndromes characterized by genome instability and cancer predisposition. As the only RecQ DNA helicase in the yeast Saccharomyces cerevisiae, Sgs1 is known to safeguard genome integrity through its role in DNA recombination. Interestingly, WRN, BLM and Sgs1 are all known to be modified by the small ubiquitin-related modifier (SUMO), although the significance of this posttranslational modification remains elusive. Here, we demonstrate that Sgs1 is specifically sumoylated under the stress of DNA double strand breaks. The major SUMO attachment site in Sgs1 is lysine 621, which lies between the Top3 binding domain and the DNA helicase domain. Surprisingly, sumoylation of K621 was found to be uniquely required for Sgs1’s role in telomere–telomere recombination. In contrast, sumoylation was dispensable for Sgs1’s roles in DNA damage tolerance, supppression of direct repeat and rDNA recombination, and promotion of top3 slow growth. Our results demonstrate that although modification by SUMO is a conserved feature of RecQ family DNA helicases, the major sites of modification are located on different domains of the protein in different organisms. We suggest that sumoylation of different domains of RecQ DNA helicases from different organisms contributes to conserved roles in regulating telomeric recombination.

PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification

Spandidos, A., Wang, X., Wang, H., Seed, B. — Wed, 11 Nov 2009 01:35:25 PST

PrimerBank (http://pga.mgh.harvard.edu/primerbank/) is a public resource for the retrieval of human and mouse primer pairs for gene expression analysis by PCR and Quantitative PCR (QPCR). A total of 306 800 primers covering most known human and mouse genes can be accessed from the PrimerBank database, together with information on these primers such as T_m, location on the transcript and amplicon size. For each gene, at least one primer pair has been designed and in many cases alternative primer pairs exist. Primers have been designed to work under the same PCR conditions, thus facilitating high-throughput QPCR. There are several ways to search for primers for the gene(s) of interest, such as by: GenBank accession number, NCBI protein accession number, NCBI gene ID, PrimerBank ID, NCBI gene symbol or gene description (keyword). In all, 26 855 primer pairs covering most known mouse genes have been experimentally validated by QPCR, agarose gel analysis, sequencing and BLAST, and all validation data can be freely accessed from the PrimerBank web site.

The Immune Epitope Database 2.0

Wed, 11 Nov 2009 01:35:22 PST

The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course of 4 years, the data from 180 978 experiments were curated manually from the literature, which covers ~99% of all publicly available information on peptide epitopes mapped in infectious agents (excluding HIV) and 93% of those mapped in allergens. In addition, data that would otherwise be unavailable to the public from 129 186 experiments were submitted directly by investigators. The curation of epitopes related to autoimmunity is expected to be completed by the end of 2010. The database can be queried by epitope structure, source organism, MHC restriction, assay type or host organism, among other criteria. The database structure, as well as its querying, browsing and reporting interfaces, was completely redesigned for the IEDB 2.0 release, which became publicly available in early 2009.

MetaBioME: a database to explore commercially useful enzymes in metagenomic datasets

Sharma, V. K., Kumar, N., Prakash, T., Taylor, T. D. — Wed, 11 Nov 2009 01:35:19 PST

Microbial enzymes have many known applications as biocatalysts in biotechnology, agriculture, medical and other industries. However, only a few enzymes are currently employed for such commercial applications. In this scenario, the current onslaught of metagenomic data provides a new unexplored treasure trove of genomic wealth that can not only enhance the enzyme repertoire by the discovery of novel commercially useful enzymes (CUEs) but can also reveal better functional variants for existing CUEs. We prepared a catalogue of CUEs using text mining of PubMed abstracts and other publicly available information, and manually curated the data to identify 510 CUEs. Further, in order to identify novel homologues of these CUEs, we identified potential ORFs in publicly available metagenomic datasets from 10 diverse sources. Using this strategy, we have developed a resource called MetaBioME (http://metasystems.riken.jp/metabiome/) that comprises (i) a database of CUEs and (ii) a comprehensive platform to facilitate homology-based computational identification of novel homologous CUEs from metagenomic and bacterial genomic datasets. Using MetaBioME, we have identified several novel homologues to known CUEs that can potentially serve as leads for further experimental verification.

Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community

Wed, 11 Nov 2009 01:36:37 PST

The Protein Structure Initiative Material Repository (PSI-MR; http://psimr.asu.edu) provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery.

Improvements to services at the European Nucleotide Archive

Wed, 11 Nov 2009 01:36:32 PST

The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe’s primary nucleotide sequence archival resource, safeguarding open nucleotide data access, engaging in worldwide collaborative data exchange and integrating with the scientific publication process. ENA has made significant contributions to the collaborative nucleotide archival arena as an active proponent of extending the traditional collaboration to cover capillary and next-generation sequencing information. We have continued to co-develop data and metadata representation formats with our collaborators for both data exchange and public data dissemination. In addition to the DDBJ/EMBL/GenBank feature table format, we share metadata formats for capillary and next-generation sequencing traces and are using and contributing to the NCBI SRA Toolkit for the long-term storage of the next-generation sequence traces. During the course of 2009, ENA has significantly improved sequence submission, search and access functionalities provided at EMBL–EBI. In this article, we briefly describe the content and scope of our archive and introduce major improvements to our services.

Processing of the Escherichia coli leuX tRNA transcript, encoding tRNALeu5, requires either the 3'->5' exoribonuclease polynucleotide phosphorylase or RNase P to remove the Rho-independent transcription terminator

Mohanty, B. K., Kushner, S. R. — Wed, 11 Nov 2009 05:54:32 PST

Here we report a unique processing pathway in Escherichia coli for tRNA^Leu5 in which the exoribonuclease polynucleotide phosphorylase (PNPase) removes the Rho-independent transcription terminator from the leuX transcript without requiring the RhlB RNA helicase. Our data demonstrate for the first time that PNPase can efficiently degrade an RNA substrate containing secondary structures in vivo. Furthermore, RNase P, an endoribonuclease that normally generates the mature 5'-ends of tRNAs, removes the leuX terminator inefficiently independent of PNPase activity. RNase P cleaves 4–7 nt downstream of the CCA determinant generating a substrate for RNase II, which removes an additional 3–4 nt. Subsequently, RNase T completes the 3' maturation process by removing the remaining 1–3 nt downstream of the CCA determinant. RNase E, G and Z are not involved in terminator removal. These results provide further evidence that the E. coli tRNA processing machinery is far more diverse than previously envisioned.

COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer

Wed, 11 Nov 2009 01:36:26 PST

The catalogue of Somatic Mutations in Cancer (COSMIC) (http://www.sanger.ac.uk/cosmic/) is the largest public resource for information on somatically acquired mutations in human cancer and is available freely without restrictions. Currently (v43, August 2009), COSMIC contains details of 1.5-million experiments performed through 13 423 genes in almost 370 000 tumours, describing over 90 000 individual mutations. Data are gathered from two sources, publications in the scientific literature, (v43 contains 7797 curated articles) and the full output of the genome-wide screens from the Cancer Genome Project (CGP) at the Sanger Institute, UK. Most of the world’s literature on point mutations in human cancer has now been curated into COSMIC and while this is continually updated, a greater emphasis on curating fusion gene mutations is driving the expansion of this information; over 2700 fusion gene mutations are now described. Whole-genome sequencing screens are now identifying large numbers of genomic rearrangements in cancer and COSMIC is now displaying details of these analyses also. Examination of COSMIC’s data is primarily web-driven, focused on providing mutation range and frequency statistics based upon a choice of gene and/or cancer phenotype. Graphical views provide easily interpretable summaries of large quantities of data, and export functions can provide precise details of user-selected data.

MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome

Serre, D., Lee, B. H., Ting, A. H. — Wed, 11 Nov 2009 05:54:11 PST

DNA methylation is an epigenetic modification involved in both normal developmental processes and disease states through the modulation of gene expression and the maintenance of genomic organization. Conventional methods of DNA methylation analysis, such as bisulfite sequencing, methylation sensitive restriction enzyme digestion and array-based detection techniques, have major limitations that impede high-throughput genome-wide analysis. We describe a novel technique, MBD-isolated Genome Sequencing (MiGS), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein and sequencing of the isolated DNA by a massively parallel sequencer. We utilized MiGS to study three isogenic cancer cell lines with varying degrees of DNA methylation. We successfully detected previously known methylated regions in these cells and identified hundreds of novel methylated regions. This technique is highly specific and sensitive and can be applied to any biological settings to identify differentially methylated regions at the genomic scale.

The tumor suppressor, parafibromin, mediates histone H3 K9 methylation for cyclin D1 repression

Yang, Y.-J., Han, J.-W., Youn, H.-D., Cho, E.-J. — Wed, 11 Nov 2009 01:36:19 PST

Parafibromin, a component of the RNA polymerase II-associated PAF1 complex, is a tumor suppressor linked to hyperparathyroidism-jaw tumor syndrome and sporadic parathyroid carcinoma. Parafibromin induces cell cycle arrest by repressing cyclin D1 via an unknown mechanism. Here, we show that parafibromin interacts with the histone methyltransferase, SUV39H1, and functions as a transcriptional repressor. The central region (128–227 amino acids) of parafibromin is important for both the interaction with SUV39H1 and transcriptional repression. Parafibromin associated with the promoter and coding regions of cyclin D1 and was required for the recruitment of SUV39H1 and the induction of H3 K9 methylation but not H3 K4 methylation. RNA interference analysis showed that SUV39H1 was critical for cyclin D1 repression. These data suggest that parafibromin plays an unexpected role as a repressor in addition to its widely known activity associated with transcriptional activation. Parafibromin as a part of the PAF1 complex might downregulate cyclin D1 expression by integrating repressive H3 K9 methylation during transcription.

The scaRNA2 is produced by an independent transcription unit and its processing is directed by the encoding region

Gerard, M.-A., Myslinski, E., Chylak, N., Baudrey, S., Krol, A., Carbon, P. — Wed, 11 Nov 2009 01:36:13 PST

The C/D box scaRNA2 is predicted to guide specific 2'-O-methylation of U2 snRNA. In contrast to other SCARNA genes, SCARNA2 appears to be independently transcribed. By transient expression of SCARNA2-reporter gene constructs, we have demonstrated that this gene is transcribed by RNA polymerase II and that the promoter elements responsible for its transcription are contained within a 161 bp region upstream of the transcription start site. In mammals, we have identified four cross species conserved promoter elements, a TATA motif, an hStaf/ZNF143 binding site and two novel elements that are required for full promoter activity. Binding of the human hStaf/ZNF143 transcription factor to its target sequence is required for promoter activity, suggesting that hStaf/ZNF143 is a fundamental regulator of the SCARNA2 gene. We also showed that RNA polymerase II continues transcription past the 3'-end of the mature RNA, irrespective of the identity of the Pol II promoter. The 3'-end processing and accumulation are governed by the sole information contained in the scaRNA2 encoding region, the maturation occurring via a particular pathway incompatible with that of mRNA or snRNA production.

Gene3D: merging structure and function for a Thousand genomes

Lees, J., Yeats, C., Redfern, O., Clegg, A., Orengo, C. — Wed, 11 Nov 2009 05:53:55 PST

Over the last 2 years the Gene3D resource has been significantly improved, and is now more accurate and with a much richer interactive display via the Gene3D website (http://gene3d.biochem.ucl.ac.uk/). Gene3D provides accurate structural domain family assignments for over 1100 genomes and nearly 10 000 000 proteins. A hidden Markov model library, constructed from the manually curated CATH structural domain hierarchy, is used to search UniProt, RefSeq and Ensembl protein sequences. The resulting matches are refined into simple multi-domain architectures using a recently developed in-house algorithm, DomainFinder 3 (available at: ftp://ftp.biochem.ucl.ac.uk/pub/gene3d_data/DomainFinder3/). The domain assignments are integrated with multiple external protein function descriptions (e.g. Gene Ontology and KEGG), structural annotations (e.g. coiled coils, disordered regions and sequence polymorphisms) and family resources (e.g. Pfam and eggNog) and displayed on the Gene3D website. The website allows users to view descriptions for both single proteins and genes and large protein sets, such as superfamilies or genomes. Subsets can then be selected for detailed investigation or associated functions and interactions can be used to expand explorations to new proteins. Gene3D also provides a set of services, including an interactive genome coverage graph visualizer, DAS annotation resources, sequence search facilities and SOAP services.

ChimerDB 2.0--a knowledgebase for fusion genes updated

Kim, P., Yoon, S., Kim, N., Lee, S., Ko, M., Lee, H., Kang, H., Kim, J., Lee, S. — Wed, 11 Nov 2009 01:36:11 PST

Chromosome translocations and gene fusions are frequent events in the human genome and have been found to cause diverse types of tumor. ChimerDB is a knowledgebase of fusion genes identified from bioinformatics analysis of transcript sequences in the GenBank and various other public resources such as the Sanger cancer genome project (CGP), OMIM, PubMed and the Mitelman’s database. In this updated version, we significantly modified the algorithm of identifying fusion transcripts. Specifically, the new algorithm is more sensitive and has detected 2699 fusion transcripts with high confidence. Furthermore, it can identify interchromosomal translocations as well as the intrachromosomal deletions or inversions of large DNA segments. Importantly, results from the analysis of next-generation sequencing data in the short read archives are incorporated as well. We updated and integrated all contents (GenBank, Sanger CGP, OMIM, PubMed publications and the Mitelman’s database), and the user-interface has been improved to support diverse types of searches and to enhance the user convenience especially in browsing PubMed articles. We also developed a new alignment viewer that should facilitate examining reliability of fusion transcripts and inferring functional significance. We expect ChimerDB 2.0, available at http://ercsb.ewha.ac.kr/fusiongene, to be a valuable tool in identifying biomarkers and drug targets.

PTGL: a database for secondary structure-based protein topologies

May, P., Kreuchwig, A., Steinke, T., Koch, I. — Wed, 11 Nov 2009 01:36:07 PST

With growing amount of experimental data, the number of known protein structures also increases continuously. Classification of protein structures helps to understand relationships between protein structure and function. The main classification methods based on secondary structures are SCOP, CATH and TOPS, which all classify under different aspects, and therefore can lead to different results. We developed a mathematically unique representation of protein structure topologies at a higher abstraction level providing new aspects of classification and enabling for a fast search through the data. Protein Topology Graph Library (PTGL; http://ptgl.zib.de) aims at providing a database on protein secondary structure topologies, including search facilities, the visualization as intuitive topology diagrams as well as in the 3D structure, and additional information. Secondary structure-based protein topologies are represented uniquely as undirected labeled graphs in four different ways allowing for exploration under different aspects. The linear notations, and the 2D and 3D diagrams of each notation facilitate a deeper understanding of protein topologies. Several search functions for topologies and sub-topologies, BLAST search possibility, and links to SCOP, CATH and PDBsum support individual and large-scale investigation of protein structures. Currently, PTGL comprises topologies of 54 859 protein structures. Main structural patterns for common structural motifs like TIM-barrel or Jelly Roll are pre-implemented, and can easily be searched.

PRGdb: a bioinformatics platform for plant resistance gene analysis

Wed, 11 Nov 2009 05:53:35 PST

PRGdb is a web accessible open-source (http://www.prgdb.org) database that represents the first bioinformatic resource providing a comprehensive overview of resistance genes (R-genes) in plants. PRGdb holds more than 16 000 known and putative R-genes belonging to 192 plant species challenged by 115 different pathogens and linked with useful biological information. The complete database includes a set of 73 manually curated reference R-genes, 6308 putative R-genes collected from NCBI and 10463 computationally predicted putative R-genes. Thanks to a user-friendly interface, data can be examined using different query tools. A home-made prediction pipeline called Disease Resistance Analysis and Gene Orthology (DRAGO), based on reference R-gene sequence data, was developed to search for plant resistance genes in public datasets such as Unigene and Genbank. New putative R-gene classes containing unknown domain combinations were discovered and characterized. The development of the PRG platform represents an important starting point to conduct various experimental tasks. The inferred cross-link between genomic and phenotypic information allows access to a large body of information to find answers to several biological questions. The database structure also permits easy integration with other data types and opens up prospects for future implementations.

A capping-independent function of MePCE in stabilizing 7SK snRNA and facilitating the assembly of 7SK snRNP

Xue, Y., Yang, Z., Chen, R., Zhou, Q. — Wed, 11 Nov 2009 01:36:05 PST

The 7SK snRNP represents a major reservoir of activity where P-TEFb, a general transcription factor key for RNA polymerase II elongation, can be withdrawn to promote gene expression, cell growth and development. Within this complex, 7SK snRNA is a central scaffold that coordinates key protein–protein interactions and maintains P-TEFb in an inactive state. Although the stability of 7SK directly affects the amount of active P-TEFb in vivo, relatively little is known about how it is maintained and how the 7SK methylphosphate capping enzyme MePCE and LARP7, a La-related protein associated with the 3'-poly(U) of 7SK, contribute to this process. Here, we show that 7SK is capped by the LARP7-free MePCE and in probably a co-transcriptional manner prior to its sequestration into 7SK snRNP. However, upon interacting with LARP7 within 7SK snRNP, MePCE loses its capping activity, probably due to the occlusion of its catalytic center by LARP7. Despite its lack of capping activity in 7SK snRNP, MePCE displays a capping-independent function to promote the LARP7–7SK interaction, which in turn stabilizes 7SK and facilitates the assembly of a stable MePCE–LARP7–7SK subcomplex. Our data indicate that MePCE and LARP7 act cooperatively to stabilize 7SK and maintain the integrity of 7SK snRNP.

Cohesin promotes the repair of ionizing radiation-induced DNA double-strand breaks in replicated chromatin

Wed, 11 Nov 2009 01:36:01 PST

The cohesin protein complex holds sister chromatids together after synthesis until mitosis. It also contributes to post-replicative DNA repair in yeast and higher eukaryotes and accumulates at sites of laser-induced damage in human cells. Our goal was to determine whether the cohesin subunits SMC1 and Rad21 contribute to DNA double-strand break repair in X-irradiated human cells in the G2 phase of the cell cycle. RNA interference-mediated depletion of SMC1 sensitized HeLa cells to X-rays. Repair of radiation-induced DNA double-strand breaks, measured by H2AX/53BP1 foci analysis, was slower in SMC1- or Rad21-depleted cells than in controls in G2 but not in G1. Inhibition of the DNA damage kinase DNA-PK, but not ATM, further inhibited foci loss in cohesin-depleted cells in G2. SMC1 depletion had no effect on DNA single-strand break repair in either G1 or late S/G2. Rad21 and SMC1 were recruited to sites of X-ray-induced DNA damage in G2-phase cells, but not in G1, and only when DNA damage was concentrated in subnuclear stripes, generated by partially shielded ultrasoft X-rays. Our results suggest that the cohesin complex contributes to cell survival by promoting the repair of radiation-induced DNA double-strand breaks in G2-phase cells in an ATM-dependent pathway.

Ensembl's 10th year

Wed, 11 Nov 2009 05:53:07 PST

Ensembl (http://www.ensembl.org) integrates genomic information for a comprehensive set of chordate genomes with a particular focus on resources for human, mouse, rat, zebrafish and other high-value sequenced genomes. We provide complete gene annotations for all supported species in addition to specific resources that target genome variation, function and evolution. Ensembl data is accessible in a variety of formats including via our genome browser, API and BioMart. This year marks the tenth anniversary of Ensembl and in that time the project has grown with advances in genome technology. As of release 56 (September 2009), Ensembl supports 51 species including marmoset, pig, zebra finch, lizard, gorilla and wallaby, which were added in the past year. Major additions and improvements to Ensembl since our previous report include the incorporation of the human GRCh37 assembly, enhanced visualisation and data-mining options for the Ensembl regulatory features and continued development of our software infrastructure.

PLANdbAffy: probe-level annotation database for Affymetrix expression microarrays

Wed, 11 Nov 2009 01:35:57 PST

Standard Affymetrix technology evaluates gene expression by measuring the intensity of mRNA hybridization with a panel of the 25-mer oligonucleotide probes, and summarizing the probe signal intensities by a robust average method. However, in many cases, signal intensity of the probe does not correlate with gene expression. This could be due to the hybridization of the probe to a transcript of another gene, mapping of the probe to an intron, alternative splicing, single nucleotide polymorphisms and other reasons. We have developed a database, PLANdbAffy (available at http://affymetrix2.bioinf.fbb.msu.ru), that contains the results of the alignment of probe sequences from five Affymetrix expression microarrays to the human genome. We have determined the probes matching the transcript-coding regions in the correct orientation. For each such probe alignment region, we determined the mRNA and EST sequences that contain the probe sequence. In the textual part of the database interface we summarize the data on the sequences that cover the probe alignment region and SNPs that are located inside it. The graphical part of our database interface is implemented as custom tracks to the UCSC genome browser that allows one to utilize all the data that are offered by UCSC browser.

The Proteomics Identifications database: 2010 update

Wed, 11 Nov 2009 01:35:53 PST

The Proteomics Identifications database (PRIDE, http://www.ebi.ac.uk/pride) at the European Bioinformatics Institute has become one of the main repositories of mass spectrometry-derived proteomics data. For the last 2 years, PRIDE data holdings have grown substantially, comprising 60 different species, more than 2.5 million protein identifications, 11.5 million peptides and over 50 million spectra by September 2009. We here describe several new and improved features in PRIDE, including the revised submission process, which now includes direct submission of fragment ion annotations. Correspondingly, it is now possible to visualize spectrum fragmentation annotations on tandem mass spectra, a key feature for compliance with journal data submission requirements. We also describe recent developments in the PRIDE BioMart interface, which now allows integrative queries that can join PRIDE data to a growing number of biological resources such as Reactome, Ensembl, InterPro and UniProt. This ability to perform extremely powerful across-domain queries will certainly be a cornerstone of future bioinformatics analyses. Finally, we highlight the importance of data sharing in the proteomics field, and the corresponding integration of PRIDE with other databases in the ProteomExchange consortium.

ComSin: database of protein structures in bound (complex) and unbound (single) states in relation to their intrinsic disorder

Wed, 11 Nov 2009 01:35:49 PST

Most of the proteins in a cell assemble into complexes to carry out their function. In this work, we have created a new database (named ComSin) of protein structures in bound (complex) and unbound (single) states to provide a researcher with exhaustive information on structures of the same or homologous proteins in bound and unbound states. From the complete Protein Data Bank (PDB), we selected 24 910 pairs of protein structures in bound and unbound states, and identified regions of intrinsic disorder. For 2448 pairs, the proteins in bound and unbound states are identical, while 7129 pairs have sequence identity 90% or larger. The developed server enables one to search for proteins in bound and unbound states with several options including sequence similarity between the corresponding proteins in bound and unbound states, and validation of interaction interfaces of protein complexes. Besides that, through our web server, one can obtain necessary information for studying disorder-to-order and order-to-disorder transitions upon complex formation, and analyze structural differences between proteins in bound and unbound states. The database is available at http://antares.protres.ru/comsin/.

3DNALandscapes: a database for exploring the conformational features of DNA

Zheng, G., Colasanti, A. V., Lu, X.-J., Olson, W. K. — Wed, 11 Nov 2009 01:35:46 PST

3DNALandscapes, located at: http://3DNAscapes.rutgers.edu, is a new database for exploring the conformational features of DNA. In contrast to most structural databases, which archive the Cartesian coordinates and/or derived parameters and images for individual structures, 3DNALandscapes enables searches of conformational information across multiple structures. The database contains a wide variety of structural parameters and molecular images, computed with the 3DNA software package and known to be useful for characterizing and understanding the sequence-dependent spatial arrangements of the DNA sugar-phosphate backbone, sugar-base side groups, base pairs, base-pair steps, groove structure, etc. The data comprise all DNA-containing structures—both free and bound to proteins, drugs and other ligands—currently available in the Protein Data Bank. The web interface allows the user to link, report, plot and analyze this information from numerous perspectives and thereby gain insight into DNA conformation, deformability and interactions in different sequence and structural contexts. The data accumulated from known, well-resolved DNA structures can serve as useful benchmarks for the analysis and simulation of new structures. The collective data can also help to understand how DNA deforms in response to proteins and other molecules and undergoes conformational rearrangements.

Network of Cancer Genes: a web resource to analyze duplicability, orthology and network properties of cancer genes

Syed, A. S., D'Antonio, M., Ciccarelli, F. D. — Wed, 11 Nov 2009 05:52:38 PST

The Network of Cancer Genes (NCG) collects and integrates data on 736 human genes that are mutated in various types of cancer. For each gene, NCG provides information on duplicability, orthology, evolutionary appearance and topological properties of the encoded protein in a comprehensive version of the human protein-protein interaction network. NCG also stores information on all primary interactors of cancer proteins, thus providing a complete overview of 5357 proteins that constitute direct and indirect determinants of human cancer. With the constant delivery of results from the mutational screenings of cancer genomes, NCG represents a versatile resource for retrieving detailed information on particular cancer genes, as well as for identifying common properties of precompiled lists of cancer genes. NCG is freely available at: http://bio.ifom-ieo-campus.it/ncg.

High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA

Nozinovic, S., Furtig, B., Jonker, H. R. A., Richter, C., Schwalbe, H. — Wed, 11 Nov 2009 01:35:44 PST

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2'-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.

Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition

Nakamura, A., Sheppard, K., Yamane, J., Yao, M., Soll, D., Tanaka, I. — Wed, 11 Nov 2009 01:35:41 PST

In many prokaryotes the biosynthesis of the amide aminoacyl-tRNAs, Gln-tRNA^Gln and Asn-tRNA^Asn, proceeds by an indirect route in which mischarged Glu-tRNA^Gln or Asp-tRNA^Asn is amidated to the correct aminoacyl-tRNA catalyzed by a tRNA-dependent amidotransferase (AdT). Two types of AdTs exist: bacteria, archaea and organelles possess heterotrimeric GatCAB, while heterodimeric GatDE occurs exclusively in archaea. Bacterial GatCAB and GatDE recognize the first base pair of the acceptor stem and the D-loop of their tRNA substrates, while archaeal GatCAB recognizes the tertiary core of the tRNA, but not the first base pair. Here, we present the crystal structure of the full-length Staphylococcus aureus GatCAB. Its GatB tail domain possesses a conserved Lys rich motif that is situated close to the variable loop in a GatCAB:tRNA^Gln docking model. This motif is also conserved in the tail domain of archaeal GatCAB, suggesting this basic region may recognize the tRNA variable loop to discriminate Asp-tRNA^Asn from Asp-tRNA^Asp in archaea. Furthermore, we identified a 3₁₀ turn in GatB that permits the bacterial GatCAB to distinguish a U1–A72 base pair from a G1–C72 pair; the absence of this element in archaeal GatCAB enables the latter enzyme to recognize aminoacyl-tRNAs with G1–C72 base pairs.

Integrative analysis of the human cis-antisense gene pairs, miRNAs and their transcription regulation patterns

Grinchuk, O. V., Jenjaroenpun, P., Orlov, Y. L., Zhou, J., Kuznetsov, V. A. — Wed, 11 Nov 2009 01:35:38 PST

Cis-antisense gene pairs (CASGPs) can transcribe mRNAs from an opposite strand of a given locus. To classify and understand diverse CASGP phenomena in the human we compiled a genome-wide catalog of CASGPs and integrated these sequences with microarray, SAGE and miRNA data. Using the concept of overlapping regions and clustering of SA transcripts by chromosome coordinates, we identified up to 9000 overlapping antisense loci. Four thousand three hundred and seventy-four of these CASGPs form 1759 complex gene architectures. We found that ~35% (6347/18160) of RefSeq genes are overlapped with the antisense transcripts. About 30% of Affymetrix U133 microarray initial sequences map transcripts of ~35% CASGPs and reveal mostly concordant expression in CASGPs. We found strong significant overrepresentation of human miRNA genes in loci of CASGPs. We developed a data-driven model of cross-talk between co-expressed CASGPs and DICER1-mediated miRNA pathway in normal spermatogenesis and in severe teratozoospermia. Specifically, we revealed complex SA structural–functional gene module composing the protein-coding genes, WDR6, DALRD3, NDUFAF3 and ncRNA precursors, mir-425 and mir-191, which could provide downregulation of ncRNA pathway via direct targeting DICER1 and basonuclin 2 transcripts by mir-425 and mir-191 in normal spermatogenesis, but this mechanism is switched off in severe teratozoospermia. The database is available from http://globalisland.bii.a-star.edu.sg/~jiangtao/sas/index3.php?link =about

JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles

Wed, 11 Nov 2009 01:35:34 PST

JASPAR (http://jaspar.genereg.net) is the leading open-access database of matrix profiles describing the DNA-binding patterns of transcription factors (TFs) and other proteins interacting with DNA in a sequence-specific manner. Its fourth major release is the largest expansion of the core database to date: the database now holds 457 non-redundant, curated profiles. The new entries include the first batch of profiles derived from ChIP-seq and ChIP-chip whole-genome binding experiments, and 177 yeast TF binding profiles. The introduction of a yeast division brings the convenience of JASPAR to an active research community. As binding models are refined by newer data, the JASPAR database now uses versioning of matrices: in this release, 12% of the older models were updated to improved versions. Classification of TF families has been improved by adopting a new DNA-binding domain nomenclature. A curated catalog of mammalian TFs is provided, extending the use of the JASPAR profiles to additional TFs belonging to the same structural family. The changes in the database set the system ready for more rapid acquisition of new high-throughput data sources. Additionally, three new special collections provide matrix profile data produced by recent alternative high-throughput approaches.

SIMAP--a comprehensive database of pre-calculated protein sequence similarities, domains, annotations and clusters

Wed, 11 Nov 2009 01:35:30 PST

The prediction of protein function as well as the reconstruction of evolutionary genesis employing sequence comparison at large is still the most powerful tool in sequence analysis. Due to the exponential growth of the number of known protein sequences and the subsequent quadratic growth of the similarity matrix, the computation of the Similarity Matrix of Proteins (SIMAP) becomes a computational intensive task. The SIMAP database provides a comprehensive and up-to-date pre-calculation of the protein sequence similarity matrix, sequence-based features and sequence clusters. As of September 2009, SIMAP covers 48 million proteins and more than 23 million non-redundant sequences. Novel features of SIMAP include the expansion of the sequence space by including databases such as ENSEMBL as well as the integration of metagenomes based on their consistent processing and annotation. Furthermore, protein function predictions by Blast2GO are pre-calculated for all sequences in SIMAP and the data access and query functions have been improved. SIMAP assists biologists to query the up-to-date sequence space systematically and facilitates large-scale downstream projects in computational biology. Access to SIMAP is freely provided through the web portal for individuals (http://mips.gsf.de/simap/) and for programmatic access through DAS (http://webclu.bio.wzw.tum.de/das/) and Web-Service (http://mips.gsf.de/webservices/services/SimapService2.0?wsdl).

MBGD update 2010: toward a comprehensive resource for exploring microbial genome diversity

Uchiyama, I., Higuchi, T., Kawai, M. — Wed, 11 Nov 2009 01:35:15 PST

The microbial genome database (MBGD) for comparative analysis is a platform for microbial comparative genomics based on automated ortholog group identification. A prominent feature of MBGD is that it allows users to create ortholog groups using a specified subgroup of organisms. The database is constantly updated and now contains almost 1000 genomes. To utilize the MBGD database as a comprehensive resource for investigating microbial genome diversity, we have developed the following advanced functionalities: (i) enhanced assignment of functional annotation, including external database links to each orthologous group, (ii) interface for choosing a set of genomes to compare based on phenotypic properties, (iii) the addition of more eukaryotic microbial genomes (fungi and protists) and some higher eukaryotes as references and (iv) enhancement of the MyMBGD mode, which allows users to add their own genomes to MBGD and now accepts raw genomic sequences without any annotation (in such a case, it runs a gene-finding procedure before identifying the orthologs). Some analysis functions, such as the function to find orthologs with similar phylogenetic patterns, have also been improved. MBGD is accessible at http://mbgd.genome.ad.jp/.

ISbrowser: an extension of ISfinder for visualizing insertion sequences in prokaryotic genomes

Kichenaradja, P., Siguier, P., Perochon, J., Chandler, M. — Wed, 11 Nov 2009 05:52:09 PST

Insertion sequences (ISs) are among the smallest and simplest autonomous transposable elements. ISfinder (http://www-is.biotoul.fr/) is a dedicated IS database which assigns names to individual ISs to maintain a coherent nomenclature, an IS repository including >3000 individual ISs from both bacteria and archaea and provides a basis for IS classification. Each IS is indexed in ISfinder with various associated pieces of information (the complete nucleotide sequence, the sequence of the ends and target sites, potential open reading frames, strain of origin, distribution in other strains and available bibliography) and classified into a group or family to provide some insight into its phylogeny. ISfinder also includes extensive background information on ISs and transposons in general. Online tools are gradually being added. At present, it is difficult to visualize the global distribution of ISs in a given bacterial genome. Such information would facilitate understanding of the impact of these small transposable elements on shaping their host genome. Here we describe ISbrowser (http://www-genome.biotoul.fr/ISbrowser.php), an extension to the ISfinder platform and a tool which permits visualization of the position, orientation and distribution of complete and partial ISs in individual prokaryotic genomes.

The UCSC genome browser database: update 2010

Wed, 11 Nov 2009 01:35:12 PST

The University of California, Santa Cruz (UCSC) Genome Browser website (http://genome.ucsc.edu/) provides a large database of publicly available sequence and annotation data along with an integrated tool set for examining and comparing the genomes of organisms, aligning sequence to genomes, and displaying and sharing users’ own annotation data. As of September 2009, genomic sequence and a basic set of annotation ‘tracks’ are provided for 47 organisms, including 14 mammals, 10 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms and a yeast. New data highlights this year include an updated human genome browser, a 44-species multiple sequence alignment track, improved variation and phenotype tracks and 16 new genome-wide ENCODE tracks. New features include drag-and-zoom navigation, a Wiki track for user-added annotations, new custom track formats for large datasets (bigBed and bigWig), a new multiple alignment output tool, links to variation and protein structure tools, in silico PCR utility enhancements, and improved track configuration tools.

Gene Expression Atlas at the European Bioinformatics Institute

Wed, 11 Nov 2009 01:35:09 PST

The Gene Expression Atlas (http://www.ebi.ac.uk/gxa) is an added-value database providing information about gene expression in different cell types, organism parts, developmental stages, disease states, sample treatments and other biological/experimental conditions. The content of this database derives from curation, re-annotation and statistical analysis of selected data from the ArrayExpress Archive of Functional Genomics Data. A simple interface allows the user to query for differential gene expression either (i) by gene names or attributes such as Gene Ontology terms, or (ii) by biological conditions, e.g. diseases, organism parts or cell types. The gene queries return the conditions where expression has been reported, while condition queries return which genes are reported to be expressed in these conditions. A combination of both query types is possible. The query results are ranked using various statistical measures and by how many independent studies in the database show the particular gene-condition association. Currently, the database contains information about more than 200 000 genes from nine species and almost 4500 biological conditions studied in over 30 000 assays from over 1000 independent studies.

Specific local induction of DNA strand breaks by infrared multi-photon absorption

Trautlein, D., Deibler, M., Leitenstorfer, A., Ferrando-May, E. — Wed, 11 Nov 2009 01:35:06 PST

Highly confined DNA damage by femtosecond laser irradiation currently arises as a powerful tool to understand DNA repair in live cells as a function of space and time. However, the specificity with respect to damage type is limited. Here, we present an irradiation procedure based on a widely tunable Er/Yb : fiber femtosecond laser source that favors the formation of DNA strand breaks over that of UV photoproducts by more than one order of magnitude. We explain this selectivity with the different power dependence of the reactions generating strand breaks, mainly involving reactive radical intermediates, and the direct photochemical process leading to UV-photoproducts. Thus, localized multi-photon excitation with a wavelength longer than 1 µm allows for the selective production of DNA strand breaks at sub-micrometer spatial resolution in the absence of photosensitizers.

Origin-dependent initiation of DNA replication within telomeric sequences

Kurth, I., Gautier, J. — Wed, 11 Nov 2009 01:35:03 PST

Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.

Molecular mechanisms of RNA polymerase--the F/E (RPB4/7) complex is required for high processivity in vitro

Hirtreiter, A., Grohmann, D., Werner, F. — Wed, 11 Nov 2009 01:35:01 PST

Transcription elongation in vitro is affected by the interactions between RNA polymerase (RNAP) subunits and the nucleic acid scaffold of the ternary elongation complex (TEC, RNAP-DNA–RNA). We have investigated the role of the RNAP subunits F/E (homologous to eukaryotic RPB4/7) during transcription elongation and termination using a wholly recombinant archaeal RNAP and synthetic nucleic acid scaffolds. The F/E complex greatly stimulates the processivity of RNAP, it enhances the formation of full length products, reduces pausing, and increases transcription termination facilitated by weak termination signals. Mutant variants of F/E that are defective in RNA binding show that these activities correlate with the nucleic acid binding properties of F/E. However, a second RNA-binding independent component also contributes to the stimulatory activities of F/E. In summary, our results suggest that interactions between RNAP subunits F/E and the RNA transcript are pivotal to the molecular mechanisms of RNAP during transcription elongation and termination.

Patrocles: a database of polymorphic miRNA-mediated gene regulation in vertebrates

Hiard, S., Charlier, C., Coppieters, W., Georges, M., Baurain, D. — Wed, 11 Nov 2009 01:34:59 PST

The Patrocles database (http://www.patrocles.org/) compiles DNA sequence polymorphisms (DSPs) that are predicted to perturb miRNA-mediated gene regulation. Distinctive features include: (i) the coverage of seven vertebrate species in its present release, aiming for more when information becomes available, (ii) the coverage of the three compartments involved in the silencing process (i.e. targets, miRNA precursors and silencing machinery), (iii) contextual information that enables users to prioritize candidate ‘Patrocles DSPs’, including graphical information on miRNA-target coexpression and eQTL effect of genotype on target expression levels, (iv) the inclusion of Copy Number Variants and eQTL information that affect miRNA precursors as well as genes encoding components of the silencing machinery and (v) a tool (Patrocles finder) that allows the user to determine whether her favorite DSP may perturb miRNA-mediated gene regulation of custom target sequences. To support the biological relevance of Patrocles' content, we searched for signatures of selection acting on ‘Patrocles single nucleotide polymorphisms (pSNPs)’ in human and mice. As expected, we found a strong signature of purifying selection against not only SNPs that destroy conserved target sites but also against SNPs that create novel, illegitimate target sites, which is reminiscent of the Texel mutation in sheep.

MicrobesOnline: an integrated portal for comparative and functional genomics

Wed, 11 Nov 2009 05:51:53 PST

Since 2003, MicrobesOnline (http://www.microbesonline.org) has been providing a community resource for comparative and functional genome analysis. The portal includes over 1000 complete genomes of bacteria, archaea and fungi and thousands of expression microarrays from diverse organisms ranging from model organisms such as Escherichia coli and Saccharomyces cerevisiae to environmental microbes such as Desulfovibrio vulgaris and Shewanella oneidensis. To assist in annotating genes and in reconstructing their evolutionary history, MicrobesOnline includes a comparative genome browser based on phylogenetic trees for every gene family as well as a species tree. To identify co-regulated genes, MicrobesOnline can search for genes based on their expression profile, and provides tools for identifying regulatory motifs and seeing if they are conserved. MicrobesOnline also includes fast phylogenetic profile searches, comparative views of metabolic pathways, operon predictions, a workbench for sequence analysis and integration with RegTransBase and other microbial genome resources. The next update of MicrobesOnline will contain significant new functionality, including comparative analysis of metagenomic sequence data. Programmatic access to the database, along with source code and documentation, is available at http://microbesonline.org/programmers.html.

Saccharomyces Genome Database provides mutant phenotype data

Wed, 11 Nov 2009 05:51:40 PST

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is a scientific database for the molecular biology and genetics of the yeast Saccharomyces cerevisiae, which is commonly known as baker’s or budding yeast. The information in SGD includes functional annotations, mapping and sequence information, protein domains and structure, expression data, mutant phenotypes, physical and genetic interactions and the primary literature from which these data are derived. Here we describe how published phenotypes and genetic interaction data are annotated and displayed in SGD.

A novel SMC-like protein, SbcE (YhaN), is involved in DNA double-strand break repair and competence in Bacillus subtilis

Krishnamurthy, M., Tadesse, S., Rothmaier, K., Graumann, P. L. — Wed, 11 Nov 2009 01:34:56 PST

Bacillus subtilis and most Gram positive bacteria possess four SMC like proteins: SMC, SbcC, RecN and the product of the yhaN gene, termed SbcE. SbcE is most similar to SbcC but contains a unique central domain. We show that SbcE plays a role during transformation in competent cells and in DNA double-strand break (DSB) repair. The phenotypes were strongly exacerbated by the additional deletion of recN or of sbcC, suggesting that all three proteins act upstream of RecA and provide distinct avenues for presynapsis. SbcE accumulated at the cell poles in competent cells, and localized as a discrete focus on the nucleoids in 10% of growing cells. This number moderately increased after treatment with DNA damaging agents and in the absence of RecN or of SbcC. Damage-induced foci of SbcE arose early after induction of DNA damage and rarely colocalized with the replication machinery. Our work shows that SMC-like proteins in B. subtilis play roles at different subcellular sites during DNA repair. SbcC operates at breaks occurring at the replication machinery, whereas RecN and SbcE function mainly, but not exclusively, at DSBs arising elsewhere on the chromosome. In agreement with this idea, we found that RecN-YFP damage-induced assemblies also arise in the absence of ongoing replication.

GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Wed, 11 Nov 2009 01:34:53 PST

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.

Accurate recognition of cis-regulatory motifs with the correct lengths in prokaryotic genomes

Li, G., Liu, B., Xu, Y. — Wed, 11 Nov 2009 01:34:50 PST

We present a new computational method for solving a classical problem, the identification problem of cis-regulatory motifs in a given set of promoter sequences, based on one key new idea. Instead of scoring candidate motifs individually like in all the existing motif-finding programs, our method scores groups of candidate motifs with similar sequences, called motif closures, using a P-value, which has substantially improved the prediction reliability over the existing methods. Our new P-value scoring scheme is sequence length independent, hence allowing direct comparisons among predicted motifs with different lengths on the same footing. We have implemented this method as a Motif Recognition Computer (MREC) program, and have extensively tested MREC on both simulated and biological data from prokaryotic genomes. Our test results indicate that MREC can accurately pick out the actual motif with the correct length as the best scoring candidate for the vast majority of the cases in our test set. We compared our prediction results with two motif-finding programs Cosmo and MEME, and found that MREC outperforms both programs across all the test cases by a large margin. The MREC program is available at http://csbl.bmb.uga.edu/~bingqiang/MREC1/.

eggNOG v2.0: extending the evolutionary genealogy of genes with enhanced non-supervised orthologous groups, species and functional annotations

Mon, 09 Nov 2009 02:15:04 PST

The identification of orthologous relationships forms the basis for most comparative genomics studies. Here, we present the second version of the eggNOG database, which contains orthologous groups (OGs) constructed through identification of reciprocal best BLAST matches and triangular linkage clustering. We applied this procedure to 630 complete genomes (529 bacteria, 46 archaea and 55 eukaryotes), which is a 2-fold increase relative to the previous version. The pipeline yielded 224 847 OGs, including 9724 extended versions of the original COG and KOG. We computed OGs for different levels of the tree of life; in addition to the species groups included in our first release (i.e. fungi, metazoa, insects, vertebrates and mammals), we have now constructed OGs for archaea, fishes, rodents and primates. We automatically annotate the non-supervised orthologous groups (NOGs) with functional descriptions, protein domains, and functional categories as defined initially for the COG/KOG database. In-depth analysis is facilitated by precomputed high-quality multiple sequence alignments and maximum-likelihood trees for each of the available OGs. Altogether, eggNOG covers 2 242 035 proteins (built from 2 590 259 proteins) and provides a broad functional description for at least 1 966 709 (88%) of them. Users can access the complete set of orthologous groups via a web interface at: http://eggnog.embl.de.

HLungDB: an integrated database of human lung cancer research

Wang, L., Xiong, Y., Sun, Y., Fang, Z., Li, L., Ji, H., Shi, T. — Mon, 09 Nov 2009 02:14:55 PST

The human lung cancer database (HLungDB) is a database with the integration of the lung cancer-related genes, proteins and miRNAs together with the corresponding clinical information. The main purpose of this platform is to establish a network of lung cancer-related molecules and to facilitate the mechanistic study of lung carcinogenesis. The entries describing the relationships between molecules and human lung cancer in the current release were extracted manually from literatures. Currently, we have collected 2585 genes and 212 miRNA with the experimental evidences involved in the different stages of lung carcinogenesis through text mining. Furthermore, we have incorporated the results from analysis of transcription factor-binding motifs, the promoters and the SNP sites for each gene. Since epigenetic alterations also play an important role in lung carcinogenesis, genes with epigenetic regulation were also included. We hope HLungDB will enrich our knowledge about lung cancer biology and eventually lead to the development of novel therapeutic strategies. HLungDB can be freely accessed at http://www.megabionet.org/bio/hlung.

GWIDD: Genome-wide protein docking database

Kundrotas, P. J., Zhu, Z., Vakser, I. A. — Mon, 09 Nov 2009 02:14:51 PST

Structural information on interacting proteins is important for understanding life processes at the molecular level. Genome-wide docking database is an integrated resource for structural studies of protein–protein interactions on the genome scale, which combines the available experimental data with models obtained by docking techniques. Current database version (August 2009) contains 25 559 experimental and modeled 3D structures for 771 organisms spanned over the entire universe of life from viruses to humans. Data are organized in a relational database with user-friendly search interface allowing exploration of the database content by a number of parameters. Search results can be interactively previewed and downloaded as PDB-formatted files, along with the information relevant to the specified interactions. The resource is freely available at http://gwidd.bioinformatics.ku.edu.

The MiST2 database: a comprehensive genomics resource on microbial signal transduction

Ulrich, L. E., Zhulin, I. B. — Mon, 09 Nov 2009 02:14:49 PST

The MiST2 database (http://mistdb.com) identifies and catalogs the repertoire of signal transduction proteins in microbial genomes. Signal transduction systems regulate the majority of cellular activities including the metabolism, development, host-recognition, biofilm production, virulence, and antibiotic resistance of human pathogens. Thus, knowledge of the proteins and interactions that comprise these communication networks is an essential component to furthering biomedical discovery. These are identified by searching protein sequences for specific domain profiles that implicate a protein in signal transduction. Compared to the previous version of the database, MiST2 contains a host of new features and improvements including the following: draft genomes; extracytoplasmic function (ECF) sigma factor protein identification; enhanced classification of signaling proteins; novel, high-quality domain models for identifying histidine kinases and response regulators; neighboring two-component genes; gene cart; better search capabilities; enhanced taxonomy browser; advanced genome browser; and a modern, biologist-friendly web interface. MiST2 currently contains 966 complete and 157 draft bacterial and archaeal genomes, which collectively contain more than 245 000 signal transduction proteins. The majority (66%) of these are one-component systems, followed by two-component proteins (26%), chemotaxis (6%), and finally ECF factors (2%).

dbDEPC: a database of Differentially Expressed Proteins in human Cancers

Li, H., He, Y., Ding, G., Wang, C., Xie, L., Li, Y. — Mon, 09 Nov 2009 02:14:44 PST

Cancer-related investigations have long been in the limelight of biomedical research. Years of effort from scientists and doctors worldwide have generated large amounts of data at the genome, transcriptome, proteome and even metabolome level, and DNA and RNA cancer signature databases have been established. Here we present a database of differentially expressed proteins in human cancers (dbDEPC), with the goal of collecting curated cancer proteomics data, providing a resource for information on protein-level expression changes, and exploring protein profile differences among different cancers. dbDEPC currently contains 1803 proteins differentially expressed in 15 cancers, curated from 65 mass spectrometry (MS) experiments in peer-reviewed publications. In addition to MS experiments, low-throughput experiment data from the same literatures and cancer-associated genes from external databases were also integrated to provide some validation information. Furthermore, dbDEPC associates differential proteins with important structural variations in the human genome, such as copy number variations or single nucleotide polymorphisms, which might be helpful for explaining changes in protein expression at the DNA level. Data in dbDEPC can be queried by protein identifier, description or sequence; the retrieved protein entry provides the differential expression pattern seen in cancers, along with detailed annotations. dbDEPC is expected to be a reference database for cancer signatures at the protein level. This database is provided at http://dbdepc.biosino.org/index/.

phiSITE: database of gene regulation in bacteriophages

Klucar, L., Stano, M., Hajduk, M. — Mon, 09 Nov 2009 02:14:41 PST

We have developed phiSITE, database of gene regulation in bacteriophages. To date it contains detailed information about more than 700 experimentally confirmed or predicted regulatory elements (promoters, operators, terminators and attachment sites) from 32 bacteriophages belonging to Siphoviridae, Myoviridae and Podoviridae families. The database is manually curated, the data are collected mainly form scientific papers, cross-referenced with other database resources (EMBL, UniProt, NCBI taxonomy database, NCBI Genome, ICTVdb, PubMed Central) and stored in SQL based database system. The system provides full text search for regulatory elements, graphical visualization of phage genomes and several export options. In addition, visualizations of gene regulatory networks for five phages (Bacillus phage GA-1, Enterobacteria phage lambda, Enterobacteria phage Mu, Enterobacteria phage P2 and Mycoplasma phage P1) have been defined and made available. The phiSITE is accessible at http://www.phisite.org/.

DNA topoisomerase I inhibition by camptothecin induces escape of RNA polymerase II from promoter-proximal pause site, antisense transcription and histone acetylation at the human HIF-1{alpha} gene locus

Baranello, L., Bertozzi, D., Fogli, M. V., Pommier, Y., Capranico, G. — Mon, 09 Nov 2009 02:14:39 PST

Top1 inhibition by camptothecin (CPT) perturbs RNA polymerase II (Pol II) density at promoters and along transcribed genes suggesting an involvement of Top1 in Pol II pausing. Here, we demonstrate that Top1 inhibition favors Pol II escape from a promoter-proximal pausing site of the human HIF-1 gene in living cells. Interestingly, alternative splicing at exon 11 was markedly altered in nascent HIF-1 mRNAs, and chromatin structure was also affected with enhanced histone acetylation and reduced nucleosome density in a manner dependent on cdk activity. Moreover, CPT increases transcription of a novel long RNA (5'aHIF1), antisense to human HIF-1 mRNA, and a known antisense RNA at the 3'-end of the gene, while decreasing mRNA levels under normoxic and hypoxic conditions. The effects require Top1, but are independent from Top1-induced replicative DNA damage. Chromatin RNA immunoprecipitation results showed that CPT can activate antisense transcription mediated by cyclin-dependent kinase (cdk) activity. Thus, Top1 inhibition can trigger a transcriptional stress, involving antisense transcription and increased chromatin accessibility, which is dependent on cdk activity and deregulated Pol II pausing. A changed balance of antisense transcripts and mRNAs may then lead to altered regulation of HIF-1 activity in human cancer cells.

MINT, the molecular interaction database: 2009 update

Fri, 06 Nov 2009 05:44:51 PST

MINT (http://mint.bio.uniroma2.it/mint) is a public repository for molecular interactions reported in peer-reviewed journals. Since its last report, MINT has grown considerably in size and evolved in scope to meet the requirements of its users. The main changes include a more precise definition of the curation policy and the development of an enhanced and user-friendly interface to facilitate the analysis of the ever-growing interaction dataset. MINT has adopted the PSI-MI standards for the annotation and for the representation of molecular interactions and is a member of the IMEx consortium.

STITCH 2: an interaction network database for small molecules and proteins

Fri, 06 Nov 2009 05:44:48 PST

Over the last years, the publicly available knowledge on interactions between small molecules and proteins has been steadily increasing. To create a network of interactions, STITCH aims to integrate the data dispersed over the literature and various databases of biological pathways, drug–target relationships and binding affinities. In STITCH 2, the number of relevant interactions is increased by incorporation of BindingDB, PharmGKB and the Comparative Toxicogenomics Database. The resulting network can be explored interactively or used as the basis for large-scale analyses. To facilitate links to other chemical databases, we adopt InChIKeys that allow identification of chemicals with a short, checksum-like string. STITCH 2.0 connects proteins from 630 organisms to over 74 000 different chemicals, including 2200 drugs. STITCH can be accessed at http://stitch.embl.de/.

T3DB: a comprehensively annotated database of common toxins and their targets

Fri, 06 Nov 2009 05:44:46 PST

In an effort to capture meaningful biological, chemical and mechanistic information about clinically relevant, commonly encountered or important toxins, we have developed the Toxin and Toxin-Target Database (T3DB). The T3DB is a unique bioinformatics resource that compiles comprehensive information about common or ubiquitous toxins and their toxin-targets into a single electronic repository. The database currently contains over 2900 small molecule and peptide toxins, 1300 toxin-targets and more than 33 000 toxin-target associations. Each T3DB record (ToxCard) contains over 80 data fields providing detailed information on chemical properties and descriptors, toxicity values, protein and gene sequences (for both targets and toxins), molecular and cellular interaction data, toxicological data, mechanistic information and references. This information has been manually extracted and manually verified from numerous sources, including other electronic databases, government documents, textbooks and scientific journals. A key focus of the T3DB is on providing ‘depth’ over ‘breadth’ with detailed descriptions, mechanisms of action, and information on toxins and toxin-targets. T3DB is fully searchable and supports extensive text, sequence, chemical structure and relational query searches, similar to those found in the Human Metabolome Database (HMDB) and DrugBank. Potential applications of the T3DB include clinical metabolomics, toxin target prediction, toxicity prediction and toxicology education. The T3DB is available online at http://www.t3db.org.

A homogeneous method for investigation of methylation-dependent protein-protein interactions in epigenetics

Fri, 06 Nov 2009 05:44:43 PST

Methylation of lysine residues on the tails of histone proteins is a major determinant of the transcription state of associated DNA coding regions. The interplay among methylation states and other histone modifications to direct transcriptional outcome is referred to as the histone code. In addition to histone methyltransferases and demethylases which function to modify the methylation state of lysine sidechains, other proteins recognize specific histone methylation marks essentially serving as code readers. While these interactions are highly specific with respect to site and methylation state of particular lysine residues, they are generally weak and therefore difficult to monitor by traditional assay techniques. Herein, we present the design and implementation of a homogeneous, miniaturizable, and sensitive assay for histone methylation-dependent interactions. We use AlphaScreen, a chemiluminescence-based technique, to monitor the interactions of chromodomains (MPP8, HP1β and CHD1), tudor domains (JMJD2A) and plant homeodomains (RAG2) with their cognate trimethyllysine histone partners. The utility of the method was demonstrated by profiling the binding specificities of chromo- and tudor domains toward several histone marks. The simplicity of design and the sensitive and robust nature of this assay should make it applicable to a range of epigenetic studies, including the search for novel inhibitors of methylation-dependent interactions.

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

Fri, 06 Nov 2009 05:44:40 PST

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA.

MEROPS: the peptidase database

Rawlings, N. D., Barrett, A. J., Bateman, A. — Thu, 05 Nov 2009 07:09:05 PST

Peptidases, their substrates and inhibitors are of great relevance to biology, medicine and biotechnology. The MEROPS database (http://merops.sanger.ac.uk) aims to fulfil the need for an integrated source of information about these. The database has a hierarchical classification in which homologous sets of peptidases and protein inhibitors are grouped into protein species, which are grouped into families, which are in turn grouped into clans. The classification framework is used for attaching information at each level. An important focus of the database has become distinguishing one peptidase from another through identifying the specificity of the peptidase in terms of where it will cleave substrates and with which inhibitors it will interact. We have collected over 39 000 known cleavage sites in proteins, peptides and synthetic substrates. These allow us to display peptidase specificity and alignments of protein substrates to give an indication of how well a cleavage site is conserved, and thus its probable physiological relevance. While the number of new peptidase families and clans has only grown slowly the number of complete genomes has greatly increased. This has allowed us to add an analysis tool to the relevant species pages to show significant gains and losses of peptidase genes relative to related species.

HHMD: the human histone modification database

Zhang, Y., Lv, J., Liu, H., Zhu, J., Su, J., Wu, Q., Qi, Y., Wang, F., Li, X. — Thu, 05 Nov 2009 07:09:02 PST

Histone modifications play important roles in chromatin remodeling, gene transcriptional regulation, stem cell maintenance and differentiation. Alterations in histone modifications may be linked to human diseases especially cancer. Histone modifications including methylation, acetylation and ubiquitylation probed by ChIP-seq, ChIP-chip and qChIP have become widely available. Mining and integration of histone modification data can be beneficial to novel biological discoveries. There has been no comprehensive data repository that is exclusive for human histone modifications. Therefore, we developed a relatively comprehensive database for human histone modifications. Human Histone Modification Database (HHMD, http://bioinfo.hrbmu.edu.cn/hhmd) focuses on the storage and integration of histone modification datasets that were obtained from laboratory experiments. The latest release of HHMD incorporates 43 location-specific histone modifications in human. To facilitate data extraction, flexible search options are built in HHMD. It can be searched by histone modification, gene ID, functional categories, chromosome location and cancer name. HHMD also includes a user-friendly visualization tool named HisModView, by which genome-wide histone modification map can be shown. HisModView facilitates the acquisition and visualization of histone modifications. The database also has manually curated information of histone modification dysregulation in nine human cancers.

PeroxisomeDB 2.0: an integrative view of the global peroxisomal metabolome

Schluter, A., Real-Chicharro, A., Gabaldon, T., Sanchez-Jimenez, F., Pujol, A. — Thu, 05 Nov 2009 07:08:57 PST

Peroxisomes are essential organelles that play a key role in redox signalling and lipid homeostasis. They contain a highly diverse enzymatic network among different species, mirroring the varied metabolic needs of the organisms. The previous PeroxisomeDB version organized the peroxisomal proteome of humans and Saccharomyces cerevisiae based on genetic and functional information into metabolic categories with a special focus on peroxisomal disease. The new release (http://www.peroxisomeDB.org) adds peroxisomal proteins from 35 newly sequenced eukaryotic genomes including fungi, yeasts, plants and lower eukaryotes. We searched these genomes for a core ensemble of 139 peroxisomal protein families and identified 2706 putative peroxisomal protein homologs. Approximately 37% of the identified homologs contained putative peroxisome targeting signals (PTS). To help develop understanding of the evolutionary relationships among peroxisomal proteins, the new database includes phylogenetic trees for 2386 of the peroxisomal proteins. Additional new features are provided, such as a tool to capture kinetic information from Brenda, CheBI and Sabio-RK databases and more than 1400 selected bibliographic references. PeroxisomeDB 2.0 is a freely available, highly interactive functional genomics platform that offers an extensive view on the peroxisomal metabolome across lineages, thus facilitating comparative genomics and systems analysis of the organelle.

InParanoid 7: new algorithms and tools for eukaryotic orthology analysis

Thu, 05 Nov 2009 07:08:53 PST

The InParanoid project gathers proteomes of completely sequenced eukaryotic species plus Escherichia coli and calculates pairwise ortholog relationships among them. The new release 7.0 of the database has grown by an order of magnitude over the previous version and now includes 100 species and their collective 1.3 million proteins organized into 42.7 million pairwise ortholog groups. The InParanoid algorithm itself has been revised and is now both more specific and sensitive. Based on results from our recent benchmarking of low-complexity filters in homology assignment, a two-pass BLAST approach was developed that makes use of high-precision compositional score matrix adjustment, but avoids the alignment truncation that sometimes follows. We have also updated the InParanoid web site (http://InParanoid.sbc.su.se). Several features have been added, the response times have been improved and the site now sports a new, clearer look. As the number of ortholog databases has grown, it has become difficult to compare among these resources due to a lack of standardized source data and incompatible representations of ortholog relationships. To facilitate data exchange and comparisons among ortholog databases, we have developed and are making available two XML schemas: SeqXML for the input sequences and OrthoXML for the output ortholog clusters.

The comprehensive microbial resource

Thu, 05 Nov 2009 07:08:48 PST

The Comprehensive Microbial Resource or CMR (http://cmr.jcvi.org) provides a web-based central resource for the display, search and analysis of the sequence and annotation for complete and publicly available bacterial and archaeal genomes. In addition to displaying the original annotation from GenBank, the CMR makes available secondary automated structural and functional annotation across all genomes to provide consistent data types necessary for effective mining of genomic data. Precomputed homology searches are stored to allow meaningful genome comparisons. The CMR supplies users with over 50 different tools to utilize the sequence and annotation data across one or more of the 571 currently available genomes. At the gene level users can view the gene annotation and underlying evidence. Genome level information includes whole genome graphical displays, biochemical pathway maps and genome summary data. Comparative tools display analysis between genomes with homology and genome alignment tools, and searches across the accessions, annotation, and evidence assigned to all genes/genomes are available. The data and tools on the CMR aid genomic research and analysis, and the CMR is included in over 200 scientific publications. The code underlying the CMR website and the CMR database are freely available for download with no license restrictions.

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis

Thu, 05 Nov 2009 07:08:45 PST

The repair of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. In higher eukaryotes, DNA DSBs are predominantly repaired by non-homologous end joining (NHEJ), but DNA ends can also be joined by an alternative error-prone mechanism termed microhomology-mediated end joining (MMEJ). In MMEJ, the repair of DNA breaks is mediated by annealing at regions of microhomology and is always associated with deletions at the break site. In budding yeast, the Mre11/Rad5/Xrs2 complex has been demonstrated to play a role in both classical NHEJ and MMEJ, but the involvement of the analogous MRE11/RAD50/NBS1 (MRN) complex in end joining in higher eukaryotes is less certain. Here we demonstrate that in Xenopus laevis egg extracts, the MRN complex is not required for classical DNA-PK-dependent NHEJ. However, the XMRN complex is necessary for resection-based end joining of mismatched DNA ends. This XMRN-dependent end joining process is independent of the core NHEJ components Ku70 and DNA-PK, occurs with delayed kinetics relative to classical NHEJ and brings about repair at sites of microhomology. These data indicate a role for the X. laevis MRN complex in MMEJ.

An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomes

Jex, A. R., Hall, R. S., Littlewood, D. T. J., Gasser, R. B. — Thu, 05 Nov 2009 07:08:42 PST

Mitochondrial (mt) genomics represents an understudied but important field of molecular biology. Increasingly, mt dysfunction is being linked to a range of human diseases, including neurodegenerative disorders, diabetes and impairment of childhood development. In addition, mt genomes provide important markers for systematic, evolutionary and population genetic studies. Some technological limitations have prevented the expanded generation and utilization of mt genomic data for some groups of organisms. These obstacles most acutely impede, but are not limited to, studies requiring the determination of complete mt genomic data from minute amounts of material (e.g. biopsy samples or microscopic organisms). Furthermore, post-sequencing bioinformatic annotation and analyses of mt genomes are time consuming and inefficient. Herein, we describe a high-throughput sequencing and bioinformatic pipeline for mt genomics, which will have implications for the annotation and analysis of other organellar (e.g. plastid or apicoplast genomes) and virus genomes as well as long, contiguous regions in nuclear genomes. We utilize this pipeline to sequence and annotate the complete mt genomes of 12 species of parasitic nematode (order Strongylida) simultaneously, each from an individual organism. These mt genomic data provide a rich source of markers for studies of the systematics and population genetics of a group of socioeconomically important pathogens of humans and other animals.

Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-thymine glycol lesion and structure of the cis-(5R,6S) thymine glycol epimer in the 5'-GTgG-3' sequence: destabilization of two base pairs at the lesion site

Brown, K. L., Roginskaya, M., Zou, Y., Altamirano, A., Basu, A. K., Stone, M. P. — Thu, 05 Nov 2009 07:08:39 PST

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson–Crick alignment. The Tg N3H imine and A N⁶ amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G•C base pair is disrupted. The solvent accessible surface and T₂ relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH₃ group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3'-neighbor guanine alleviate steric clash with the 5'-neighbor base pair. Tg also destabilizes the 5'-neighbor G•C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

Optimizing nucleotide sequence ensembles for combinatorial protein libraries using a genetic algorithm

Craig, R. A., Lu, J., Luo, J., Shi, L., Liao, L. — Wed, 04 Nov 2009 04:39:17 PST

Protein libraries are essential to the field of protein engineering. Increasingly, probabilistic protein design is being used to synthesize combinatorial protein libraries, which allow the protein engineer to explore a vast space of amino acid sequences, while at the same time placing restrictions on the amino acid distributions. To this end, if site-specific amino acid probabilities are input as the target, then the codon nucleotide distributions that match this target distribution can be used to generate a partially randomized gene library. However, it turns out to be a highly nontrivial computational task to find the codon nucleotide distributions that exactly matches a given target distribution of amino acids. We first showed that for any given target distribution an exact solution may not exist at all. Formulated as a constrained optimization problem, we then developed a genetic algorithm-based approach to find codon nucleotide distributions that match as closely as possible to the target amino acid distribution. As compared with the previous gradient descent method on various objective functions, the new method consistently gave more optimized distributions as measured by the relative entropy between the calculated and the target distributions. To simulate the actual lab solutions, new objective functions were designed to allow for two separate sets of codons in seeking a better match to the target amino acid distribution.

The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

Wed, 04 Nov 2009 04:39:11 PST

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m⁷GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

Non-redundant patent sequence databases with value-added annotations at two levels

Sun, 01 Nov 2009 23:30:34 PST

The European Bioinformatics Institute (EMBL-EBI) provides public access to patent data, including abstracts, chemical compounds and sequences. Sequences can appear multiple times due to the filing of the same invention with multiple patent offices, or the use of the same sequence by different inventors in different contexts. Information relating to the source invention may be incomplete, and biological information available in patent documents elsewhere may not be reflected in the annotation of the sequence. Search and analysis of these data have become increasingly challenging for both the scientific and intellectual-property communities. Here, we report a collection of non-redundant patent sequence databases, which cover the EMBL-Bank nucleotides patent class and the patent protein databases and contain value-added annotations from patent documents. The databases were created at two levels by the use of sequence MD5 checksums. Sequences within a level-1 cluster are 100% identical over their whole length. Level-2 clusters were defined by sub-grouping level-1 clusters based on patent family information. Value-added annotations, such as publication number corrections, earliest publication dates and feature collations, significantly enhance the quality of the data, allowing for better tracking and cross-referencing. The databases are available format: http://www.ebi.ac.uk/patentdata/nr/.

Xenbase: gene expression and improved integration

Sun, 01 Nov 2009 23:30:30 PST

Xenbase (www.xenbase.org), the model organism database for Xenopus laevis and X. (Silurana) tropicalis, is the principal centralized resource of genomic, development data and community information for Xenopus research. Recent improvements include the addition of the literature and interaction tabs to gene catalog pages. New content has been added including a section on gene expression patterns that incorporates image data from the literature, large scale screens and community submissions. Gene expression data are integrated into the gene catalog via an expression tab and is also searchable by multiple criteria using an expression search interface. The gene catalog has grown to contain over 15 000 genes. Collaboration with the European Xenopus Research Center (EXRC) has resulted in a stock center section with data on frog lines supplied by the EXRC. Numerous improvements have also been made to search and navigation. Xenbase is also the source of the Xenopus Anatomical Ontology and the clearinghouse for Xenopus gene nomenclature.

CORUM: the comprehensive resource of mammalian protein complexes--2009

Sun, 01 Nov 2009 23:30:27 PST

CORUM is a database that provides a manually curated repository of experimentally characterized protein complexes from mammalian organisms, mainly human (64%), mouse (16%) and rat (12%). Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. The new CORUM 2.0 release encompasses 2837 protein complexes offering the largest and most comprehensive publicly available dataset of mammalian protein complexes. The CORUM dataset is built from 3198 different genes, representing ~16% of the protein coding genes in humans. Each protein complex is described by a protein complex name, subunit composition, function as well as the literature reference that characterizes the respective protein complex. Recent developments include mapping of functional annotation to Gene Ontology terms as well as cross-references to Entrez Gene identifiers. In addition, a ‘Phylogenetic Conservation’ analysis tool was implemented that analyses the potential occurrence of orthologous protein complex subunits in mammals and other selected groups of organisms. This allows one to predict the occurrence of protein complexes in different phylogenetic groups. CORUM is freely accessible at (http://mips.helmholtz-muenchen.de/genre/proj/corum/index.html).

FlyTF: improved annotation and enhanced functionality of the Drosophila transcription factor database

Sun, 01 Nov 2009 23:30:24 PST

FlyTF (http://www.flytf.org) is a database of computationally predicted and/or experimentally verified site-specific transcription factors (TFs) in the fruit fly Drosophila melanogaster. The manual classification of TFs in the initial version of FlyTF that concentrated primarily on the DNA-binding characteristics of the proteins has now been extended to a more fine-grained annotation of both DNA binding and regulatory properties in the new release. Furthermore, experimental evidence from the literature was classified into a defined vocabulary, and in collaboration with FlyBase, translated into Gene Ontology (GO) annotation. While our GO annotations will also be available through FlyBase as they will be incorporated into the genes’ official GO annotation in the future, the entire evidence used for classification including computational predictions and quotes from the literature can be accessed through FlyTF. The FlyTF website now builds upon the InterMine framework, which provides experimental and computational biologists with powerful search and filter functionality, list management tools and access to genomic information associated with the TFs.

RegPrecise: a database of curated genomic inferences of transcriptional regulatory interactions in prokaryotes

Sun, 01 Nov 2009 23:30:21 PST

The RegPrecise database (http://regprecise.lbl.gov) was developed for capturing, visualization and analysis of predicted transcription factor regulons in prokaryotes that were reconstructed and manually curated by utilizing the comparative genomic approach. A significant number of high-quality inferences of transcriptional regulatory interactions have been already accumulated for diverse taxonomic groups of bacteria. The reconstructed regulons include transcription factors, their cognate DNA motifs and regulated genes/operons linked to the candidate transcription factor binding sites. The RegPrecise allows for browsing the regulon collections for: (i) conservation of DNA binding sites and regulated genes for a particular regulon across diverse taxonomic lineages; (ii) sets of regulons for a family of transcription factors; (iii) repertoire of regulons in a particular taxonomic group of species; (iv) regulons associated with a metabolic pathway or a biological process in various genomes. The initial release of the database includes ~11 500 candidate binding sites for ~400 orthologous groups of transcription factors from over 350 prokaryotic genomes. Majority of these data are represented by genome-wide regulon reconstructions in Shewanella and Streptococcus genera and a large-scale prediction of regulons for the LacI family of transcription factors. Another section in the database represents the results of accurate regulon propagation to the closely related genomes.

Ensembl genomes: Extending ensembl across the taxonomic space

Sun, 01 Nov 2009 23:30:16 PST

Ensembl Genomes (http://www.ensemblgenomes.org) is a new portal offering integrated access to genome-scale data from non-vertebrate species of scientific interest, developed using the Ensembl genome annotation and visualisation platform. Ensembl Genomes consists of five sub-portals (for bacteria, protists, fungi, plants and invertebrate metazoa) designed to complement the availability of vertebrate genomes in Ensembl. Many of the databases supporting the portal have been built in close collaboration with the scientific community, which we consider as essential for maintaining the accuracy and usefulness of the resource. A common set of user interfaces (which include a graphical genome browser, FTP, BLAST search, a query optimised data warehouse, programmatic access, and a Perl API) is provided for all domains. Data types incorporated include annotation of (protein and non-protein coding) genes, cross references to external resources, and high throughput experimental data (e.g. data from large scale studies of gene expression and polymorphism visualised in their genomic context). Additionally, extensive comparative analysis has been performed, both within defined clades and across the wider taxonomy, and sequence alignments and gene trees resulting from this can be accessed through the site.

CyanoBase: the cyanobacteria genome database update 2010

Fri, 30 Oct 2009 05:57:11 PDT

CyanoBase (http://genome.kazusa.or.jp/cyanobase) is the genome database for cyanobacteria, which are model organisms for photosynthesis. The database houses cyanobacteria species information, complete genome sequences, genome-scale experiment data, gene information, gene annotations and mutant information. In this version, we updated these datasets and improved the navigation and the visual display of the data views. In addition, a web service API now enables users to retrieve the data in various formats with other tools, seamlessly.

UTRdb and UTRsite (RELEASE 2010): a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs

Fri, 30 Oct 2009 05:57:05 PDT

The 5' and 3' untranslated regions of eukaryotic mRNAs (UTRs) play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization and message stability. UTRdb is a curated database of 5' and 3' untranslated sequences of eukaryotic mRNAs, derived from several sources of primary data. Experimentally validated functional motifs are annotated and also collated as the UTRsite database where more specific information on the functional motifs and cross-links to interacting regulatory protein are provided. In the current update, the UTR entries have been organized in a gene-centric structure to better visualize and retrieve 5' and 3'UTR variants generated by alternative initiation and termination of transcription and alternative splicing. Experimentally validated miRNA targets and conserved sequence elements are also annotated. The integration of UTRdb with genomic data has allowed the implementation of an efficient annotation system and a powerful retrieval resource for the selection and extraction of specific UTR subsets. All internet resources implemented for retrieval and functional analysis of 5' and 3' untranslated regions of eukaryotic mRNAs are accessible at http://utrdb.ba.itb.cnr.it/.

fPOP: footprinting functional pockets of proteins by comparative spatial patterns

Tseng, Y. Y., Chen, Z. J., Li, W.-H. — Fri, 30 Oct 2009 05:57:01 PDT

fPOP (footprinting Pockets Of Proteins, http://pocket.uchicago.edu/fpop/) is a relational database of the protein functional surfaces identified by analyzing the shapes of binding sites in ~42 700 structures, including both holo and apo forms. We previously used a purely geometric method to extract the spatial patterns of functional surfaces (split pockets) in ~19 000 bound structures and constructed a database, SplitPocket (http://pocket.uchicago.edu/). These functional surfaces are now used as spatial templates to predict the binding surfaces of unbound structures. To conduct a shape comparison, we use the Smith–Waterman algorithm to footprint an unbound pocket fragment with those of the functional surfaces in SplitPocket. The pairwise alignment of the unbound and bound pocket fragments is used to evaluate the local structural similarity via geometric matching. The final results of our large-scale computation, including ~90 000 identified or predicted functional surfaces, are stored in fPOP. This database provides an easily accessible resource for studying functional surfaces, assessing conformational changes between bound and unbound forms and analyzing functional divergence. Moreover, it may facilitate the exploration of the physicochemical textures of molecules and the inference of protein function. Finally, our approach provides a framework for classification of proteins into families on the basis of their functional surfaces.

KEGG for representation and analysis of molecular networks involving diseases and drugs

Kanehisa, M., Goto, S., Furumichi, M., Tanabe, M., Hirakawa, M. — Fri, 30 Oct 2009 05:56:56 PDT

Most human diseases are complex multi-factorial diseases resulting from the combination of various genetic and environmental factors. In the KEGG database resource (http://www.genome.jp/kegg/), diseases are viewed as perturbed states of the molecular system, and drugs as perturbants to the molecular system. Disease information is computerized in two forms: pathway maps and gene/molecule lists. The KEGG PATHWAY database contains pathway maps for the molecular systems in both normal and perturbed states. In the KEGG DISEASE database, each disease is represented by a list of known disease genes, any known environmental factors at the molecular level, diagnostic markers and therapeutic drugs, which may reflect the underlying molecular system. The KEGG DRUG database contains chemical structures and/or chemical components of all drugs in Japan, including crude drugs and TCM (Traditional Chinese Medicine) formulas, and drugs in the USA and Europe. This database also captures knowledge about two types of molecular networks: the interaction network with target molecules, metabolizing enzymes, other drugs, etc. and the chemical structure transformation network in the history of drug development. The new disease/drug information resource named KEGG MEDICUS can be used as a reference knowledge base for computational analysis of molecular networks, especially, by integrating large-scale experimental datasets.

PepX: a structural database of non-redundant protein-peptide complexes

Fri, 30 Oct 2009 05:56:52 PDT

Although protein–peptide interactions are estimated to constitute up to 40% of all protein interactions, relatively little information is available for the structural details of these interactions. Peptide-mediated interactions are a prime target for drug design because they are predominantly present in signaling and regulatory networks. A reliable data set of nonredundant protein–peptide complexes is indispensable as a basis for modeling and design, but current data sets for protein–peptide interactions are often biased towards specific types of interactions or are limited to interactions with small ligands. In PepX (http://pepx.switchlab.org), we have designed an unbiased and exhaustive data set of all protein–peptide complexes available in the Protein Data Bank with peptide lengths up to 35 residues. In addition, these complexes have been clustered based on their binding interfaces rather than sequence homology, providing a set of structurally diverse protein–peptide interactions. The final data set contains 505 unique protein–peptide interface clusters from 1431 complexes. Thorough annotation of each complex with both biological and structural information facilitates searching for and browsing through individual complexes and clusters. Moreover, we provide an additional source of data for peptide design by annotating peptides with naturally occurring backbone variations using fragment clusters from the BriX database.

NNDB: the nearest neighbor parameter database for predicting stability of nucleic acid secondary structure

Turner, D. H., Mathews, D. H. — Fri, 30 Oct 2009 05:56:47 PDT

The Nearest Neighbor Database (NNDB, http://rna.urmc.rochester.edu/NNDB) is a web-based resource for disseminating parameter sets for predicting nucleic acid secondary structure stabilities. For each set of parameters, the database includes the set of rules with descriptive text, sequence-dependent parameters in plain text and html, literature references to experiments and usage tutorials. The initial release covers parameters for predicting RNA folding free energy and enthalpy changes.

Comparative analyses of time-course gene expression profiles of the long-lived sch9{Delta} mutant

Ge, H., Wei, M., Fabrizio, P., Hu, J., Cheng, C., Longo, V. D., Li, L. M. — Fri, 30 Oct 2009 05:56:43 PDT

In an attempt to elucidate the underlying longevity-promoting mechanisms of mutants lacking SCH9, which live three times as long as wild type chronologically, we measured their time-course gene expression profiles. We interpreted their expression time differences by statistical inferences based on prior biological knowledge, and identified the following significant changes: (i) between 12 and 24 h, stress response genes were up-regulated by larger fold changes and ribosomal RNA (rRNA) processing genes were down-regulated more dramatically; (ii) mitochondrial ribosomal protein genes were not up-regulated between 12 and 60 h as wild type were; (iii) electron transport, oxidative phosphorylation and TCA genes were down-regulated early; (iv) the up-regulation of TCA and electron transport was accompanied by deep down-regulation of rRNA processing over time; and (v) rRNA processing genes were more volatile over time, and three associated cis-regulatory elements [rRNA processing element (rRPE), polymerase A and C (PAC) and glucose response element (GRE)] were identified. Deletion of AZF1, which encodes the transcriptional factor that binds to the GRE element, reversed the lifespan extension of sch9. The significant alterations in these time-dependent expression profiles imply that the lack of SCH9 turns on the longevity programme that extends the lifespan through changes in metabolic pathways and protection mechanisms, particularly, the regulation of aerobic respiration and rRNA processing.

DRYGIN: a database of quantitative genetic interaction networks in yeast

Fri, 30 Oct 2009 05:56:35 PDT

Genetic interactions are highly informative for deciphering the underlying functional principles that govern how genes control cell processes. Recent developments in Synthetic Genetic Array (SGA) analysis enable the mapping of quantitative genetic interactions on a genome-wide scale. To facilitate access to this resource, which will ultimately represent a complete genetic interaction network for a eukaryotic cell, we developed DRYGIN (Data Resource of Yeast Genetic Interactions)—a web database system that aims at providing a central platform for yeast genetic network analysis and visualization. In addition to providing an interface for searching the SGA genetic interactions, DRYGIN also integrates other data sources, in order to associate the genetic interactions with pathway information, protein complexes, other binary genetic and physical interactions, and Gene Ontology functional annotation. DRYGIN version 1.0 currently holds more than 6 million measurements of genetic interacting pairs involving ~4500 genes, and is available at http://drygin.ccbr.utoronto.ca

PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update

Fri, 30 Oct 2009 05:56:31 PDT

The PhosPhAt database of Arabidopsis phosphorylation sites was initially launched in August 2007. Since then, along with 10-fold increase in database entries, functionality of PhosPhAt (phosphat.mpimp-golm.mpg.de) has been considerably upgraded and re-designed. PhosPhAt is now more of a web application with the inclusion of advanced search functions allowing combinatorial searches by Boolean terms. The results output now includes interactive visualization of annotated fragmentation spectra and the ability to export spectra and peptide sequences as text files for use in other applications. We have also implemented dynamic links to other web resources thus augmenting PhosPhAt-specific information with external protein-related data. For experimental phosphorylation sites with information about dynamic behavior in response to external stimuli, we display simple time-resolved diagrams. We have included predictions for pT and pY sites and updated pS predictions. Access to prediction algorithm now allows ‘on-the-fly’ prediction of phosphorylation of any user-uploaded protein sequence. Protein Pfam domain structures are now mapped onto the protein sequence display next to experimental and predicted phosphorylation sites. Finally, we have implemented functional annotation of proteins using MAPMAN ontology. These new developments make the PhosPhAt resource a useful and powerful tool for the scientific community as a whole beyond the plant sciences.

The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

Thu, 29 Oct 2009 02:10:52 PDT

An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the ~30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8.

Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples

Thu, 29 Oct 2009 02:10:49 PDT

Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

IPD--the Immuno Polymorphism Database

Robinson, J., Mistry, K., McWilliam, H., Lopez, R., Marsh, S. G. E. — Thu, 29 Oct 2009 02:10:46 PDT

The Immuno Polymorphism Database (IPD) (http://www.ebi.ac.uk/ipd/) is a set of specialist databases related to the study of polymorphic genes in the immune system. The IPD project works with specialist groups or nomenclature committees who provide and curate individual sections before they are submitted to IPD for online publication. The IPD project stores all the data in a set of related databases. IPD currently consists of four databases: IPD-KIR, contains the allelic sequences of Killer-cell Immunoglobulin-like Receptors, IPD-MHC, is a database of sequences of the Major Histocompatibility Complex of different species; IPD-human platelet antigens, alloantigens expressed only on platelets and IPD-ESTDAB, which provides access to the European Searchable Tumour cell-line database, a cell bank of immunologically characterised melanoma cell lines. The data is currently available online from the website and ftp directory.

Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase

Rajendra, E., Venkitaraman, A. R. — Thu, 29 Oct 2009 02:10:43 PDT

The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approximately 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC–RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.

Single-cell transcriptional analysis of taste sensory neuron pair in Caenorhabditis elegans

Takayama, J., Faumont, S., Kunitomo, H., Lockery, S. R., Iino, Y. — Thu, 29 Oct 2009 02:10:40 PDT

The nervous system is composed of a wide variety of neurons. A description of the transcriptional profiles of each neuron would yield enormous information about the molecular mechanisms that define morphological or functional characteristics. Here we show that RNA isolation from single neurons is feasible by using an optimized mRNA tagging method. This method extracts transcripts in the target cells by co-immunoprecipitation of the complexes of RNA and epitope-tagged poly(A) binding protein expressed specifically in the cells. With this method and genome-wide microarray, we compared the transcriptional profiles of two functionally different neurons in the main C. elegans gustatory neuron class ASE. Eight of the 13 known subtype-specific genes were successfully detected. Additionally, we identified nine novel genes including a receptor guanylyl cyclase, secreted proteins, a TRPC channel and uncharacterized genes conserved among nematodes, suggesting the two neurons are substantially different than previously thought. The expression of these novel genes was controlled by the previously known regulatory network for subtype differentiation. We also describe unique motif organization within individual gene groups classified by the expression patterns in ASE. Our study paves the way to the complete catalog of the expression profiles of individual C. elegans neurons.

Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance

Wed, 28 Oct 2009 04:44:49 PDT

The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3'-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

Wed, 28 Oct 2009 04:44:40 PDT

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

Wed, 28 Oct 2009 04:44:36 PDT

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.

The integrated microbial genomes system: an expanding comparative analysis resource

Wed, 28 Oct 2009 04:44:32 PDT

The integrated microbial genomes (IMG) system serves as a community resource for comparative analysis of publicly available genomes in a comprehensive integrated context. IMG contains both draft and complete microbial genomes integrated with other publicly available genomes from all three domains of life, together with a large number of plasmids and viruses. IMG provides tools and viewers for analyzing and reviewing the annotations of genes and genomes in a comparative context. Since its first release in 2005, IMG’s data content and analytical capabilities have been constantly expanded through regular releases. Several companion IMG systems have been set up in order to serve domain specific needs, such as expert review of genome annotations. IMG is available at http://img.jgi.doe.gov.

Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana

Wed, 28 Oct 2009 04:44:29 PDT

The nuclear cap-binding protein complex (CBC) participates in 5' splice site selection of introns that are proximal to the mRNA cap. However, it is not known whether CBC has a role in alternative splicing. Using an RT–PCR alternative splicing panel, we analysed 435 alternative splicing events in Arabidopsis thaliana genes, encoding mainly transcription factors, splicing factors and stress-related proteins. Splicing profiles were determined in wild type plants, the cbp20 and cbp80(abh1) single mutants and the cbp20/80 double mutant. The alternative splicing events included alternative 5' and 3' splice site selection, exon skipping and intron retention. Significant changes in the ratios of alternative splicing isoforms were found in 101 genes. Of these, 41% were common to all three CBC mutants and 15% were observed only in the double mutant. The cbp80(abh1) and cbp20/80 mutants had many more changes in alternative splicing in common than did cbp20 and cbp20/80 suggesting that CBP80 plays a more significant role in alternative splicing than CBP20, probably being a platform for interactions with other splicing factors. Cap-binding proteins and the CBC are therefore directly involved in alternative splicing of some Arabidopsis genes and in most cases influenced alternative splicing of the first intron, particularly at the 5' splice site.

Expression of stress-response ATF3 is mediated by Nrf2 in astrocytes

Kim, K.-H., Jeong, J.-Y., Surh, Y.-J., Kim, K.-W. — Wed, 28 Oct 2009 04:44:23 PDT

Activating Transcription Factor 3 (ATF3), a member of the ATF/CREB family, is induced rapidly by various stresses. Its induction mechanism and role in response to changes in cellular redox status, however, have not been elucidated. Here, we found that NF-E2-related factor 2 (Nrf2), a transcription factor known to bind to antioxidant response element (ARE) in promoters, transcriptionally upregulated ATF3 expression in astrocytes. Treatment with Nrf2 activators and oxidants provoked ATF3 induction in astrocytes, whereas its expression was reduced in Nrf2-depleted cells. We further demonstrated that the consensus ARE in the ATF3 promoter is critical for Nrf2-mediation by promoter analyses using an ATF3 promoter-driven luciferase construct and a chromatin immunoprecipitation assay. In addition, we found that Nrf2-dependent ATF3 induction contributed to the antioxidative and cytoprotective functions of Nrf2 in astrocytes. Taken together, our findings suggest that ATF3 is a new target for Nrf2 and has a cytoprotective function in astrocytes.

Preferential access to genetic information from endogenous hominin ancient DNA and accurate quantitative SNP-typing via SPEX

Brotherton, P., Sanchez, J. J., Cooper, A., Endicott, P. — Tue, 27 Oct 2009 21:28:30 PDT

The analysis of targeted genetic loci from ancient, forensic and clinical samples is usually built upon polymerase chain reaction (PCR)-generated sequence data. However, many studies have shown that PCR amplification from poor-quality DNA templates can create sequence artefacts at significant levels. With hominin (human and other hominid) samples, the pervasive presence of highly PCR-amplifiable human DNA contaminants in the vast majority of samples can lead to the creation of recombinant hybrids and other non-authentic artefacts. The resulting PCR-generated sequences can then be difficult, if not impossible, to authenticate. In contrast, single primer extension (SPEX)-based approaches can genotype single nucleotide polymorphisms from ancient fragments of DNA as accurately as modern DNA. A single SPEX-type assay can amplify just one of the duplex DNA strands at target loci and generate a multi-fold depth-of-coverage, with non-authentic recombinant hybrids reduced to undetectable levels. Crucially, SPEX-type approaches can preferentially access genetic information from damaged and degraded endogenous ancient DNA templates over modern human DNA contaminants. The development of SPEX-type assays offers the potential for highly accurate, quantitative genotyping from ancient hominin samples.

The Mouse Genome Database: enhancements and updates

Tue, 27 Oct 2009 21:28:22 PDT

The Mouse Genome Database (MGD) is a major component of the Mouse Genome Informatics (MGI, http://www.informatics.jax.org/) database resource and serves as the primary community model organism database for the laboratory mouse. MGD is the authoritative source for mouse gene, allele and strain nomenclature and for phenotype and functional annotations of mouse genes. MGD contains comprehensive data and information related to mouse genes and their functions, standardized descriptions of mouse phenotypes, extensive integration of DNA and protein sequence data, normalized representation of genome and genome variant information including comparative data on mammalian genes. Data for MGD are obtained from diverse sources including manual curation of the biomedical literature and direct contributions from individual investigator’s laboratories and major informatics resource centers, such as Ensembl, UniProt and NCBI. MGD collaborates with the bioinformatics community on the development and use of biomedical ontologies such as the Gene Ontology and the Mammalian Phenotype Ontology. Recent improvements in MGD described here includes integration of mouse gene trap allele and sequence data, integration of gene targeting information from the International Knockout Mouse Consortium, deployment of an MGI Biomart, and enhancements to our batch query capability for customized data access and retrieval.

Megx.net: integrated database resource for marine ecological genomics

Sun, 25 Oct 2009 22:13:23 PDT

Megx.net is a database and portal that provides integrated access to georeferenced marker genes, environment data and marine genome and metagenome projects for microbial ecological genomics. All data are stored in the Microbial Ecological Genomics DataBase (MegDB), which is subdivided to hold both sequence and habitat data and global environmental data layers. The extended system provides access to several hundreds of genomes and metagenomes from prokaryotes and phages, as well as over a million small and large subunit ribosomal RNA sequences. With the refined Genes Mapserver, all data can be interactively visualized on a world map and statistics describing environmental parameters can be calculated. Sequence entries have been curated to comply with the proposed minimal standards for genomes and metagenomes (MIGS/MIMS) of the Genomic Standards Consortium. Access to data is facilitated by Web Services. The updated megx.net portal offers microbial ecologists greatly enhanced database content, and new features and tools for data analysis, all of which are freely accessible from our webpage http://www.megx.net.

PDBe: Protein Data Bank in Europe

Sun, 25 Oct 2009 22:13:20 PDT

The Protein Data Bank in Europe (PDBe) (http://www.ebi.ac.uk/pdbe/) is actively working with its Worldwide Protein Data Bank partners to enhance the quality and consistency of the international archive of bio-macromolecular structure data, the Protein Data Bank (PDB). PDBe also works closely with its collaborators at the European Bioinformatics Institute and the scientific community around the world to enhance its databases and services by adding curated and actively maintained derived data to the existing structural data in the PDB. We have developed a new database infrastructure based on the remediated PDB archive data and a specially designed database for storing information on interactions between proteins and bound molecules. The group has developed new services that allow users to carry out simple textual queries or more complex 3D structure-based queries. The newly designed ‘PDBeView Atlas pages’ provide an overview of an individual PDB entry in a user-friendly layout and serve as a starting point to further explore the information available in the PDBe database. PDBe’s active involvement with the X-ray crystallography, Nuclear Magnetic Resonance spectroscopy and cryo-Electron Microscopy communities have resulted in improved tools for structure deposition and analysis.

Molecular crowding creates an essential environment for the formation of stable G-quadruplexes in long double-stranded DNA

Zheng, K.-w., Chen, Z., Hao, Y.-h., Tan, Z. — Sun, 25 Oct 2009 22:13:16 PDT

Large numbers of guanine-rich sequences with potential to form G-quadruplexes have been identified in genomes of various organisms. Such sequences are constrained at both ends by long DNA duplex with a complementary strand in close proximity to compete for duplex formation. G-quadruplex/duplex competition in long double-stranded DNA has rarely been studied. In this work, we used DMS footprinting and gel electrophoresis to study G-quadruplex formation in long double-stranded DNA derived from human genome under both dilute and molecular crowding condition created by PEG. G-quadruplex formation was observed in the process of RNA transcription and after heat denaturation/renaturation under molecular crowding condition. Our results showed that the heat denaturation/renaturation treatment followed by gel electrophoresis could provide a simple method to quantitatively access the ability of G-quadruplex formation in long double-stranded DNA. The effect of K⁺ and PEG concentration was investigated and we found that stable G-quadruplexes could only form under the crowding condition with PEG at concentrations near the physiological concentration of biomass in living cells. This observation reveals a physical basis for the formation of stable G-quadruplexes in genome and supports its presence under the in vivo molecular crowding condition.

Transposition of the human Hsmar1 transposon: rate-limiting steps and the importance of the flanking TA dinucleotide in second strand cleavage

Claeys Bouuaert, C., Chalmers, R. — Sun, 25 Oct 2009 22:13:07 PDT

Hsmar1 is a member of the mariner family of DNA transposons. Although widespread in nature, their molecular mechanism remains obscure. Many other cut-and-paste elements use a hairpin intermediate to cleave the two strands of DNA at each transposon end. However, this intermediate is absent in mariner, suggesting that these elements use a fundamentally different mechanism for second-strand cleavage. We have taken advantage of the faithful and efficient in vitro reaction provided by Hsmar1 to characterize the products and intermediates of transposition. We report different factors that particularly affect the reaction, which are the reaction pH and the transposase concentration. Kinetic analysis revealed that first-strand nicking and integration are rapid. The rate of the reaction is limited in part by the divalent metal ion-dependent assembly of a complex between transposase and the transposon end(s) prior to the first catalytic step. Second-strand cleavage is the rate-limiting catalytic step of the reaction. We discuss our data in light of a model for the two metal ion catalytic mechanism and propose that mariner excision involves a significant conformational change between first- and second-strand cleavage at each transposon end. Furthermore, this conformational change requires specific contacts between transposase and the flanking TA dinucleotide.

PROSITE, a protein domain database for functional characterization and annotation

Sun, 25 Oct 2009 22:13:03 PDT

PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 (~70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/.

Martini: using literature keywords to compare gene sets

Sun, 25 Oct 2009 22:13:00 PDT

Life scientists are often interested to compare two gene sets to gain insight into differences between two distinct, but related, phenotypes or conditions. Several tools have been developed for comparing gene sets, most of which find Gene Ontology (GO) terms that are significantly over-represented in one gene set. However, such tools often return GO terms that are too generic or too few to be informative. Here, we present Martini, an easy-to-use tool for comparing gene sets. Martini is based, not on GO, but on keywords extracted from Medline abstracts; Martini also supports a much wider range of species than comparable tools. To evaluate Martini we created a benchmark based on the human cell cycle, and we tested several comparable tools (CoPub, FatiGO, Marmite and ProfCom). Martini had the best benchmark performance, delivering a more detailed and accurate description of function. Martini also gave best or equal performance with three other datasets (related to Arabidopsis, melanoma and ovarian cancer), suggesting that Martini represents an advance in the automated comparison of gene sets. In agreement with previous studies, our results further suggest that literature-derived keywords are a richer source of gene-function information than GO annotations. Martini is freely available at http://martini.embl.de.

Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

Basenko, E. Y., Cesare, A. J., Iyer, S., Griffith, J. D., McEachern, M. J. — Sun, 25 Oct 2009 22:12:55 PDT

Some human cancers maintain their telomeres using the alternative lengthening of telomeres (ALT) mechanism; a process thought to involve recombination. Different types of recombinational telomere elongation pathways have been identified in yeasts. In senescing yeast telomerase deletion (ter1-) mutants with very short telomeres, it has been hypothesized that copying a tiny telomeric circle (t-circle) by a rolling circle mechanism is the key event in telomere elongation. In other cases more closely resembling ALT cells, such as the stn1-M1 mutant of Kluyveromyces lactis, the telomeres appear to be continuously unstable and routinely reach very large sizes. By employing two-dimensional gel electrophoresis and electron microscopy, we show that stn1-M1 cells contain abundant double stranded t-circles ranging from ~100 to 30 000 bp in size. We also observed small single-stranded t-circles, specifically composed of the G-rich telomeric strand and tailed circles resembling rolling circle replication intermediates. The t-circles most likely arose from recombination events that also resulted in telomere truncations. The findings strengthen the possibility that t-circles contribute to telomere maintenance in stn1-M1 and ALT cells.

PlnTFDB: updated content and new features of the plant transcription factor database

Sun, 25 Oct 2009 22:12:51 PDT

The Plant Transcription Factor Database (PlnTFDB; http://plntfdb.bio.uni-potsdam.de/v3.0/) is an integrative database that provides putatively complete sets of transcription factors (TFs) and other transcriptional regulators (TRs) in plant species (sensu lato) whose genomes have been completely sequenced and annotated. The complete sets of 84 families of TFs and TRs from 19 species ranging from unicellular red and green algae to angiosperms are included in PlnTFDB, representing >1.6 billion years of evolution of gene regulatory networks. For each gene family, a basic description is provided that is complemented by literature references, and multiple sequence alignments of protein domains. TF or TR gene entries include information of expressed sequence tags, 3D protein structures of homologous proteins, domain architecture and cross-links to other computational resources online. Moreover, the different species in PlnTFDB are linked to each other by means of orthologous genes facilitating cross-species comparisons.

BioNumbers--the database of key numbers in molecular and cell biology

Milo, R., Jorgensen, P., Moran, U., Weber, G., Springer, M. — Fri, 23 Oct 2009 08:48:50 PDT

BioNumbers (http://www.bionumbers.hms.harvard.edu) is a database of key numbers in molecular and cell biology—the quantitative properties of biological systems of interest to computational, systems and molecular cell biologists. Contents of the database range from cell sizes to metabolite concentrations, from reaction rates to generation times, from genome sizes to the number of mitochondria in a cell. While always of importance to biologists, having numbers in hand is becoming increasingly critical for experimenting, modeling, and analyzing biological systems. BioNumbers was motivated by an appreciation of how long it can take to find even the simplest number in the vast biological literature. All numbers are taken directly from a literature source and that reference is provided with the number. BioNumbers is designed to be highly searchable and queries can be performed by keywords or browsed by menus. BioNumbers is a collaborative community platform where registered users can add content and make comments on existing data. All new entries and commentary are curated to maintain high quality. Here we describe the database characteristics and implementation, demonstrate its use, and discuss future directions for its development.

Chemical Entities of Biological Interest: an update

Fri, 23 Oct 2009 08:48:47 PDT

Chemical Entities of Biological Interest (ChEBI) is a freely available dictionary of molecular entities focused on ‘small’ chemical compounds. The molecular entities in question are either natural products or synthetic products used to intervene in the processes of living organisms. Genome-encoded macromolecules (nucleic acids, proteins and peptides derived from proteins by cleavage) are not as a rule included in ChEBI. In addition to molecular entities, ChEBI contains groups (parts of molecular entities) and classes of entities. ChEBI includes an ontological classification, whereby the relationships between molecular entities or classes of entities and their parents and/or children are specified. ChEBI is available online at http://www.ebi.ac.uk/chebi/. This article reports on new features in ChEBI since the last NAR report in 2007, including substructure and similarity searching, a submission tool for authoring of ChEBI datasets by the community and a 30-fold increase in the number of chemical structures stored in ChEBI.

Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence

Harve, K. S., Lareu, R., Rajagopalan, R., Raghunath, M. — Fri, 23 Oct 2009 08:48:44 PDT

Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.

CpG_MI: a novel approach for identifying functional CpG islands in mammalian genomes

Su, J., Zhang, Y., Lv, J., Liu, H., Tang, X., Wang, F., Qi, Y., Feng, Y., Li, X. — Fri, 23 Oct 2009 08:48:36 PDT

CpG islands (CGIs) are CpG-rich regions compared to CpG-depleted bulk DNA of mammalian genomes and are generally regarded as the epigenetic regulatory regions in association with unmethylation, promoter activity and histone modifications. Accurate identification of CpG islands with epigenetic regulatory function in bulk genomes is of wide interest. Here, the common features of functional CGIs are identified using an average mutual information method to differentiate functional CGIs from the remaining CGIs. A new approach (CpG mutual information, CpG_MI) was further explored to identify functional CGIs based on the cumulative mutual information of physical distances between two neighboring CpGs. Compared to current approaches, CpG_MI achieved the highest prediction accuracy. This approach also identified new functional CGIs overlapping with gene promoter regions which were missed by other algorithms. Nearly all CGIs identified by CpG_MI overlapped with histone modification marks. CpG_MI could also be used to identify potential functional CGIs in other mammalian genomes, as the CpG dinucleotide contents and cumulative mutual information distributions are almost the same among six mammalian genomes in our analysis. It is a reliable quantitative tool for the identification of functional CGIs from bulk genomes and helps in understanding the relationships between genomic functional elements and epigenomic modifications.

Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding

Fri, 23 Oct 2009 08:48:30 PDT

The white gene, which is responsible for eye pigmentation, is widely used to study position effects in Drosophila. As a result of insertion of P-element vectors containing mini-white without enhancers into random chromosomal sites, flies with different eye color phenotypes appear, which is usually explained by the influence of positive/negative regulatory elements located around the insertion site. We found that, in more than 70% of cases when mini-white expression was subject to positive position effects, deletion of the white promoter had no effect on eye pigmentation; in these cases, the transposon was inserted into the transcribed regions of genes. Therefore, transcription through the mini-white gene could be responsible for high levels of its expression in most of chromosomal sites. Consistently with this conclusion, transcriptional terminators proved to be efficient in protecting mini-white expression from positive position effects. On the other hand, the best characterized Drosophila gypsy insulator was poorly effective in terminating transcription and, as a consequence, only partially protected mini-white expression from these effects. Thus, to ensure maximum protection of a transgene from position effects, a perfect boundary/insulator element should combine three activities: to block enhancers, to provide a barrier between active and repressed chromatin, and to terminate transcription.

'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target

Lustig, Y., Wachtel, C., Safro, M., Liu, L., Michaeli, S. — Fri, 23 Oct 2009 08:48:25 PDT

In this study we describe a novel method to investigate the RNA–RNA interactions between a small RNA and its target that we termed ‘RNA walk’. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT–PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by ‘RNA walk’ and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that ‘RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.

MouseBook: an integrated portal of mouse resources

Fri, 23 Oct 2009 08:48:22 PDT

The MouseBook (http://www.mousebook.org) databases and web portal provide access to information about mutant mouse lines held as live or cryopreserved stocks at MRC Harwell. The MouseBook portal integrates curated information from the MRC Harwell stock resource, and other Harwell databases, with information from external data resources to provide value-added information above and beyond what is available through other routes such as International Mouse Stain Resource (IMSR). MouseBook can be searched either using an intuitive Google style free text search or using the Mammalian Phenotype (MP) ontology tree structure. Text searches can be on gene, allele, strain identifier (e.g. MGI ID) or phenotype term and are assisted by automatic recognition of term types and autocompletion of gene and allele names covered by the database. Results are returned in a tabbed format providing categorized results identified from each of the catalogs in MouseBook. Individual result lines from each catalog include information on gene, allele, chromosomal location and phenotype, and provide a simple click-through link to further information as well as ordering the strain. The infrastructure underlying MouseBook has been designed to be extensible, allowing additional data sources to be added and enabling other sites to make their data directly available through MouseBook.

The NCBI BioSystems database

Fri, 23 Oct 2009 08:48:18 PDT

The NCBI BioSystems database, found at http://www.ncbi.nlm.nih.gov/biosystems/, centralizes and cross-links existing biological systems databases, increasing their utility and target audience by integrating their pathways and systems into NCBI resources. This integration allows users of NCBI’s Entrez databases to quickly categorize proteins, genes and small molecules by metabolic pathway, disease state or other BioSystem type, without requiring time-consuming inference of biological relationships from the literature or multiple experimental datasets.

MMBGX: a method for estimating expression at the isoform level and detecting differential splicing using whole-transcript Affymetrix arrays

Turro, E., Lewin, A., Rose, A., Dallman, M. J., Richardson, S. — Fri, 23 Oct 2009 08:48:14 PDT

Affymetrix has recently developed whole-transcript GeneChips—‘Gene’ and ‘Exon’ arrays—which interrogate exons along the length of each gene. Although each probe on these arrays is intended to hybridize perfectly to only one transcriptional target, many probes match multiple transcripts located in different parts of the genome or alternative isoforms of the same gene. Existing statistical methods for estimating expression do not take this into account and are thus prone to producing inflated estimates. We propose a method, Multi-Mapping Bayesian Gene eXpression (MMBGX), which disaggregates the signal at ‘multi-match’ probes. When applied to Gene arrays, MMBGX removes the upward bias of gene-level expression estimates. When applied to Exon arrays, it can further disaggregate the signal between alternative transcripts of the same gene, providing expression estimates of individual splice variants. We demonstrate the performance of MMBGX on simulated data and a tissue mixture data set. We then show that MMBGX can estimate the expression of alternative isoforms within one experimental condition, confirming our results by RT-PCR. Finally, we show that our method for detecting differential splicing has a lower error rate than standard exon-level approaches on a previously validated colon cancer data set.

FtsK translocation on DNA stops at XerCD-dif

Graham, J. E., Sivanathan, V., Sherratt, D. J., Arciszewska, L. K. — Fri, 23 Oct 2009 08:48:11 PDT

Escherichia coli FtsK is a powerful, fast, double-stranded DNA translocase, which can strip proteins from DNA. FtsK acts in the late stages of chromosome segregation by facilitating sister chromosome unlinking at the division septum. KOPS-guided DNA translocation directs FtsK towards dif, located within the replication terminus region, ter, where FtsK activates XerCD site-specific recombination. Here we show that FtsK translocation stops specifically at XerCD-dif, thereby preventing removal of XerCD from dif and allowing activation of chromosome unlinking by recombination. Stoppage of translocation at XerCD-dif is accompanied by a reduction in FtsK ATPase and is not associated with FtsK dissociation from DNA. Specific stoppage at recombinase-DNA complexes does not require the FtsK regulatory subdomain, which interacts with XerD, and is not dependent on either recombinase-mediated DNA cleavage activity, or the formation of synaptic complexes.

Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents

Fri, 23 Oct 2009 08:48:07 PDT

For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

SALAD database: a motif-based database of protein annotations for plant comparative genomics

Mihara, M., Itoh, T., Izawa, T. — Fri, 23 Oct 2009 08:48:04 PDT

Proteins often have several motifs with distinct evolutionary histories. Proteins with similar motifs have similar biochemical properties and thus related biological functions. We constructed a unique comparative genomics database termed the SALAD database (http://salad.dna.affrc.go.jp/salad/) from plant-genome-based proteome data sets. We extracted evolutionarily conserved motifs by MEME software from 209 529 protein-sequence annotation groups selected by BLASTP from the proteome data sets of 10 species: rice, sorghum, Arabidopsis thaliana, grape, a lycophyte, a moss, 3 algae, and yeast. Similarity clustering of each protein group was performed by pairwise scoring of the motif patterns of the sequences. The SALAD database provides a user-friendly graphical viewer that displays a motif pattern diagram linked to the resulting bootstrapped dendrogram for each protein group. Amino-acid-sequence-based and nucleotide-sequence-based phylogenetic trees for motif combination alignment, a logo comparison diagram for each clade in the tree, and a Pfam-domain pattern diagram are also available. We also developed a viewer named ‘SALAD on ARRAYs’ to view arbitrary microarray data sets of paralogous genes linked to the same dendrogram in a window. The SALAD database is a powerful tool for comparing protein sequences and can provide valuable hints for biological analysis.

Profiling the selectivity of DNA ligases in an array format with mass spectrometry

Kim, J., Mrksich, M. — Fri, 23 Oct 2009 08:48:01 PDT

This article describes a method for the global profiling of the substrate specificities of DNA ligases and illustrates examples using the Taq and T4 DNA ligases. The method combines oligonucleotide arrays, which offer the benefits of high throughput and multiplexed assays, with mass spectrometry to permit label-free assays of ligase activity. Arrays were prepared by immobilizing ternary biotin-tagged DNA substrates to a self-assembled monolayer presenting a layer of streptavidin protein. The array represented complexes having all possible matched and mismatched base pairs at the 3' side of the nick site and also included a number of deletions and insertions at this site. The arrays were treated with ligases and adenosine triphosphate or analogs of the nucleotide triphosphate and then analyzed by matrix-assisted laser desorption-ionization mass spectrometry to determine the yields for both adenylation of the 5'-probe strand and joining of the two probe strands. The resulting activity profiles reveal the basis for specificity of the ligases and also point to strategies that use ATP analogs to improve specificity. This work introduces a method that can be applied to profile a broad range of enzymes that operate on nucleic acid substrates.

Maximization of negative correlations in time-course gene expression data for enhancing understanding of molecular pathways

Zeng, T., Li, J. — Fri, 23 Oct 2009 08:47:58 PDT

Positive correlation can be diversely instantiated as shifting, scaling or geometric pattern, and it has been extensively explored for time-course gene expression data and pathway analysis. Recently, biological studies emerge a trend focusing on the notion of negative correlations such as opposite expression patterns, complementary patterns and self-negative regulation of transcription factors (TFs). These biological ideas and primitive observations motivate us to formulate and investigate the problem of maximizing negative correlations. The objective is to discover all maximal negative correlations of statistical and biological significance from time-course gene expression data for enhancing our understanding of molecular pathways. Given a gene expression matrix, a maximal negative correlation is defined as an activation–inhibition two-way expression pattern (AIE pattern). We propose a parameter-free algorithm to enumerate the complete set of AIE patterns from a data set. This algorithm can identify significant negative correlations that cannot be identified by the traditional clustering/biclustering methods. To demonstrate the biological usefulness of AIE patterns in the analysis of molecular pathways, we conducted deep case studies for AIE patterns identified from Yeast cell cycle data sets. In particular, in the analysis of the Lysine biosynthesis pathway, new regulation modules and pathway components were inferred according to a significant negative correlation which is likely caused by a co-regulation of the TFs at the higher layer of the biological network. We conjecture that maximal negative correlations between genes are actually a common characteristic in molecular pathways, which can provide insights into the cell stress response study, drug response evaluation, etc.

DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells

Fri, 23 Oct 2009 08:47:47 PDT

Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ~50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA->T and AL-II-dG->T transversions.

The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation

Barron, V. A., Zhu, H., Hinman, M. N., Ladd, A. N., Lou, H. — Fri, 23 Oct 2009 08:47:42 PDT

The CUG-BP and ETR-3 like factors (CELF) are a family of six highly conserved RNA-binding proteins that preferentially bind to UG-rich sequences. One of the key functions of these proteins is to mediate alternative splicing in a number of tissues, including brain, heart and muscle. To fully understand the function of CELF proteins, it is important to identify downstream targets of CELF proteins. In this communication, we report that neurofibromatosis type I (NF1) exon 23a is a novel target of CELF protein-mediated splicing regulation in neuron-like cells. NF1 regulates Ras signaling, and the isoform that excludes exon 23a shows 10 times greater ability to down-regulate Ras signaling than the isoform that includes exon 23a. Five of the six CELF proteins strongly suppress the inclusion of NF1 exon 23a. Over-expression or siRNA knockdown of these proteins in cell transfection experiments altered the levels of NF1 exon 23a inclusion. In vitro binding and splicing analyses demonstrate that CELF proteins block splicing through interfering with binding of U2AF⁶⁵. These studies, combined with our previous investigations demonstrating a role for Hu proteins and TIA-1/TIAR in controlling NF1 exon 23a inclusion, highlight the complex nature of regulation of this important alternative splicing event.

Enhanced gene repair mediated by methyl-CpG-modified single-stranded oligonucleotides

Bertoni, C., Rustagi, A., Rando, T. A. — Fri, 23 Oct 2009 08:47:39 PDT

Gene editing mediated by oligonucleotides has been shown to induce stable single base alterations in genomic DNA in both prokaryotic and eukaryotic organisms. However, the low frequencies of gene repair have limited its applicability for both basic manipulation of genomic sequences and for the development of therapeutic approaches for genetic disorders. Here, we show that single-stranded oligodeoxynucleotides (ssODNs) containing a methyl-CpG modification and capable of binding to the methyl-CpG binding domain protein 4 (MBD4) are able to induce >10-fold higher levels of gene correction than ssODNs lacking the specific modification. Correction was stably inherited through cell division and was confirmed at the protein, transcript and genomic levels. Downregulation of MBD4 expression using RNAi prevented the enhancement of gene correction efficacy obtained using the methyl-CpG-modified ssODN, demonstrating the specificity of the repair mechanism being recruited. Our data demonstrate that efficient manipulation of genomic targets can be achieved and controlled by the type of ssODN used and by modulation of the repair mechanism involved in the correction process. This new generation of ssODNs represents an important technological advance that is likely to have an impact on multiple applications, especially for gene therapy where permanent correction of the genetic defect has clear advantages over viral and other nonviral approaches currently being tested.

Fine tuning of the E. coli NusB:NusE complex affinity to BoxA RNA is required for processive antitermination

Burmann, B. M., Luo, X., Rosch, P., Wahl, M. C., Gottesman, M. E. — Fri, 23 Oct 2009 08:47:35 PDT

Phage propagation in Escherichia coli host cells requires transcription antitermination on the chromosome mediated by N protein and four host Nus factors, NusA, B, E (ribosomal S10) and G. Interaction of E. coli NusB:NusE heterodimer with the single stranded BoxA motif of nutL or nutR RNA is crucial for this reaction. Similarly, binding of NusB:NusE to a BoxA motif is essential to suppress transcription termination in the ribosomal RNA (rrn) operons. We used fluorescence anisotropy to measure the binding properties of NusB and of NusB:NusE heterodimer to BoxA-containing RNAs differing in length and sequence. Our results demonstrate that BoxA is necessary and sufficient for binding. We also studied the gain-of-function D118N NusB mutant that allows growth in nusA1 or nusE71 mutants. In vivo burst-size determinations, CD thermal unfolding measurements and X-ray crystallography of this as well as various other NusB D118 mutants showed the importance of size and polarity of amino acid 118 for RNA binding and other interactions. Our work suggests that the affinity of the NusB:NusE complex to BoxA RNA is precisely tuned to maximize control of transcription termination.

Limited complementarity between U1 snRNA and a retroviral 5' splice site permits its attenuation via RNA secondary structure

Zychlinski, D., Erkelenz, S., Melhorn, V., Baum, C., Schaal, H., Bohne, J. — Fri, 23 Oct 2009 08:47:32 PDT

Multiple types of regulation are used by cells and viruses to control alternative splicing. In murine leukemia virus, accessibility of the 5' splice site (ss) is regulated by an upstream region, which can fold into a complex RNA stem–loop structure. The underlying sequence of the structure itself is negligible, since most of it could be functionally replaced by a simple heterologous RNA stem–loop preserving the wild-type splicing pattern. Increasing the RNA duplex formation between U1 snRNA and the 5'ss by a compensatory mutation in position +6 led to enhanced splicing. Interestingly, this mutation affects splicing only in the context of the secondary structure, arguing for a dynamic interplay between structure and primary 5'ss sequence. The reduced 5'ss accessibility could also be counteracted by recruiting a splicing enhancer domain via a modified MS2 phage coat protein to a single binding site at the tip of the simple RNA stem–loop. The mechanism of 5'ss attenuation was revealed using hyperstable U1 snRNA mutants, showing that restricted U1 snRNP access is the cause of retroviral alternative splicing.

The IntAct molecular interaction database in 2010

Thu, 22 Oct 2009 08:45:17 PDT

IntAct is an open-source, open data molecular interaction database and toolkit. Data is abstracted from the literature or from direct data depositions by expert curators following a deep annotation model providing a high level of detail. As of September 2009, IntAct contains over 200.000 curated binary interaction evidences. In response to the growing data volume and user requests, IntAct now provides a two-tiered view of the interaction data. The search interface allows the user to iteratively develop complex queries, exploiting the detailed annotation with hierarchical controlled vocabularies. Results are provided at any stage in a simplified, tabular view. Specialized views then allows ‘zooming in’ on the full annotation of interactions, interactors and their properties. IntAct source code and data are freely available at http://www.ebi.ac.uk/intact.

The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases

Thu, 22 Oct 2009 08:45:14 PDT

The MetaCyc database (MetaCyc.org) is a comprehensive and freely accessible resource for metabolic pathways and enzymes from all domains of life. The pathways in MetaCyc are experimentally determined, small-molecule metabolic pathways and are curated from the primary scientific literature. With more than 1400 pathways, MetaCyc is the largest collection of metabolic pathways currently available. Pathways reactions are linked to one or more well-characterized enzymes, and both pathways and enzymes are annotated with reviews, evidence codes, and literature citations. BioCyc (BioCyc.org) is a collection of more than 500 organism-specific Pathway/Genome Databases (PGDBs). Each BioCyc PGDB contains the full genome and predicted metabolic network of one organism. The network, which is predicted by the Pathway Tools software using MetaCyc as a reference, consists of metabolites, enzymes, reactions and metabolic pathways. BioCyc PGDBs also contain additional features, such as predicted operons, transport systems, and pathway hole-fillers. The BioCyc Web site offers several tools for the analysis of the PGDBs, including Omics Viewers that enable visualization of omics datasets on two different genome-scale diagrams and tools for comparative analysis. The BioCyc PGDBs generated by SRI are offered for adoption by any party interested in curation of metabolic, regulatory, and genome-related information about an organism.

Sequence context outside the target region influences the effectiveness of miR-223 target sites in the RhoB 3'UTR

Sun, G., Li, H., Rossi, J. J. — Thu, 22 Oct 2009 08:45:10 PDT

MicroRNAs (miRNAs) are 21–22 nucleotide regulatory small RNAs that repress message translation via base-pairing with complementary sequences in the 3' untranslated region (3'UTR) of targeted transcripts. To date, it is still difficult to find a true miRNA target due to lack of a clear understanding of how miRNAs functionally interact with their targeted transcripts for efficient repression. Previous studies have shown that nucleotides 2 to 7 at the 5'-end of a mature miRNA, the ‘seed sequence’, can nucleate miRNA/target interactions. In the current study, we have validated that the RhoB mRNA is a bona fide miR-223 target. We have analyzed the functional activities of two miR223-binding sites within the RhoB 3'UTR. We find that the two miR-223 target sites in the RhoB 3'UTR contribute differentially to the total repression of RhoB translation. Moreover, we demonstrate that some AU-rich motifs located upstream of the distal miRNA-binding site enhance miRNA function, independent of the miRNA target sequences being tested. We also demonstrate that the AU-rich sequence elements are polar, and do not affect the activities of miRNAs whose sites lie upstream of these elements. These studies provide further support for the role of sequences outside of miRNA target region influencing miRNA function.

Modeling tissue-specific structural patterns in human and mouse promoters

Vandenbon, A., Nakai, K. — Thu, 22 Oct 2009 08:45:03 PDT

Sets of genes expressed in the same tissue are believed to be under the regulation of a similar set of transcription factors, and can thus be assumed to contain similar structural patterns in their regulatory regions. Here we present a study of the structural patterns in promoters of genes expressed specifically in 26 human and 34 mouse tissues. For each tissue we constructed promoter structure models, taking into account presences of motifs, their positioning to the transcription start site, and pairwise positioning of motifs. We found that 35 out of 60 models (58%) were able to distinguish positive test promoter sequences from control promoter sequences with statistical significance. Models with high performance include those for liver, skeletal muscle, kidney and tongue. Many of the important structural patterns in these models involve transcription factors of known importance in the tissues in question and structural patterns tend to be conserved between human and mouse. In addition to that, promoter models for related tissues tend to have high inter-tissue performance, indicating that their promoters share common structural patterns. Together, these results illustrate the validity of our models, but also indicate that the promoter structures for some tissues are easier to model than those of others.

NF90 selectively represses the translation of target mRNAs bearing an AU-rich signature motif

Thu, 22 Oct 2009 08:44:59 PDT

The RNA-binding protein nuclear factor 90 (NF90) has been implicated in the stabilization, transport and translational control of several target mRNAs. However, a systematic analysis of NF90 target mRNAs has not been performed. Here, we use ribonucleoprotein immunoprecipitation analysis to identify a large subset of NF90-associated mRNAs. Comparison of the 3'-untranslated regions (UTRs) of these mRNAs led to the elucidation of a 25- to 30-nucleotide, RNA signature motif rich in adenines and uracils. Insertion of the AU-rich NF90 motif (‘NF90m’) in the 3'UTR of an EGFP heterologous reporter did not affect the steady-state level of the chimeric EGFP-NF90m mRNA or its cytosolic abundance. Instead, the translation of EGFP-NF90m mRNA was specifically repressed in an NF90-dependent manner, as determined by analysing nascent EGFP translation, the distribution of chimeric mRNAs on polysome gradients and the steady-state levels of expressed EGFP protein. The interaction of endogenous NF90 with target mRNAs was validated after testing both endogenous mRNAs and recombinant biotinylated transcripts containing NF90 motif hits. Further analysis showed that the stability of endogenous NF90 target mRNAs was not significantly influenced by NF90 abundance, while their translation increased when NF90 levels were reduced. In summary, we have identified an AU-rich RNA motif present in NF90 target mRNAs and have obtained evidence that NF90 represses the translation of this subset of mRNAs.

Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs

Chen, T. S., Lai, R. C., Lee, M. M., Choo, A. B. H., Lee, C. N., Lim, S. K. — Thu, 22 Oct 2009 08:44:54 PDT

Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell–cell communication. Here we investigate if mesenchymal stem cells (MSCs) known for secreting therapeutic paracrine factors also secrete RNA-containing microparticles. We observed that human embryonic stem cell (hESC)-derived MSC conditioned medium contained small RNAs (less than 300 nt) encapsulated in cholesterol-rich phospholipid vesicles as evidenced by their RNase sensitivity only in the presence of a sodium dodecyl sulfate-based cell lysis buffer, phospholipase A2 and a chelator of cholesterol, cyclodextrin and the restoration of their lower than expected density by detergent or phospholipase A2 treatment. MicroRNAs (miRNAs) such as hsa-let-7b and hsa-let-7g were present in a high precursor (pre)- to mature miRNA ratio by microarray analysis and quantitative reverse transcription–polymerase chain reaction. The pre-miRNAs were cleaved to mature miRNA by RNase III in vitro. High performance liquid chromatography-purified RNA-containing vesicles have a hydrodynamic radius of 55–65 nm and were readily taken up by H9C2 cardiomyocytes. This study suggests that MSCs could facilitate miRNA-mediated intercellular communication by secreting microparticles enriched for pre-miRNA.

Comparing effects of mTR and mTERT deletion on gene expression and DNA damage response: a critical examination of telomere length maintenance-independent roles of telomerase

Vidal-Cardenas, S. L., Greider, C. W. — Thu, 22 Oct 2009 08:44:49 PDT

Telomerase, the essential enzyme that maintains telomere length, contains two core components, TERT and TR. Early studies in yeast and mouse showed that loss of telomerase leads to phenotypes only after several generations, due to telomere shortening. However, recent studies have suggested additional roles for telomerase components in transcription and the response to DNA damage. To examine these potential telomere length maintenance-independent roles of telomerase components, we examined first generation mTR^–/– and mTERT^–/– mice with long telomeres. We used gene expression profiling and found no genes that were differentially expressed in mTR^–/– G1 mice and mTERT^–/– G1 mice compared with wild-type mice. We also compared the response to DNA damage in mTR^–/–G1 and mTERT^–/– G1 mouse embryonic fibroblasts, and found no increase in the response to DNA damage in the absence of either telomerase component compared to wild-type. We conclude that, under physiologic conditions, neither mTR nor mTERT acts as a transcription factor or plays a role in the DNA damage response.

DDBJ launches a new archive database with analytical tools for next-generation sequence data

Thu, 22 Oct 2009 08:44:44 PDT

The DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) has collected and released 1 701 110 entries/1 116 138 614 bases between July 2008 and June 2009. A few highlighted data releases from DDBJ were the complete genome sequence of an endosymbiont within protist cells in the termite gut and Cap Analysis Gene Expression tags for human and mouse deposited from the Functional Annotation of the Mammalian cDNA consortium. In this period, we started a novel user announcement service using Really Simple Syndication (RSS) to deliver a list of data released from DDBJ on a daily basis. Comprehensive visualization of a DDBJ release data was attempted by using a word cloud program. Moreover, a new archive for sequencing data from next-generation sequencers, the ‘DDBJ Read Archive’ (DRA), was launched. Concurrently, for read data registered in DRA, a semi-automatic annotation tool called the ‘DDBJ Read Annotation Pipeline’ was released as a preliminary step. The pipeline consists of two parts: basic analysis for reference genome mapping and de novo assembly and high-level analysis of structural and functional annotations. These new services will aid users’ research and provide easier access to DDBJ databases.

GeMInA, Genomic Metadata for Infectious Agents, a geospatial surveillance pathogen database

Thu, 22 Oct 2009 08:44:36 PDT

The Gemina system (http://gemina.igs.umaryland.edu) identifies, standardizes and integrates the outbreak metadata for the breadth of NIAID category A–C viral and bacterial pathogens, thereby providing an investigative and surveillance tool describing the Who [Host], What [Disease, Symptom], When [Date], Where [Location] and How [Pathogen, Environmental Source, Reservoir, Transmission Method] for each pathogen. The Gemina database will provide a greater understanding of the interactions of viral and bacterial pathogens with their hosts and infectious diseases through in-depth literature text-mining, integrated outbreak metadata, outbreak surveillance tools, extensive ontology development, metadata curation and representative genomic sequence identification and standards development. The Gemina web interface provides metadata selection and retrieval of a pathogen's; Infection Systems (Pathogen, Host, Disease, Transmission Method and Anatomy) and Incidents (Location and Date) along with a hosts Age and Gender. The Gemina system provides an integrated investigative and geospatial surveillance system connecting pathogens, pathogen products and disease anchored on the taxonomic ID of the pathogen and host to identify the breadth of hosts and diseases known for these pathogens, to identify the extent of outbreak locations, and to identify unique genomic regions with the DNA Signature Insignia Detection Tool.

Molecular dynamics simulations and coupled nucleotide substitution experiments indicate the nature of A{middle dot}A base pairing and a putative structure of the coralyne-induced homo-adenine duplex

Joung, I. S., Persil Cetinkol, O., Hud, N. V., Cheatham, T. E. — Thu, 22 Oct 2009 08:44:28 PDT

Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson–Crick DNA. Hud’s laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine–adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the six most favorable duplex structures. The two most stable structures had trans glycosidic bonds, but distinct pairing geometries, i.e. either Watson–Crick Hoogsteen (transWH) or Watson–Crick Watson–Crick (transWW) with stability of transWH > transWW. To narrow down the possibilities, 7-deaza adenine base substitutions (dA->7) were engineered into homo-(dA) sequences. These substitutions significantly reduced the thermal stability of the coralyne-induced homo-(dA) structure. These experiments strongly suggest the involvement of N7 in the coralyne-induced A·A base pairs. Moreover, due to the differential effect on melting as a function of the location of the dA->7 mutations, these results are consistent with the N1–N7 base pairing of the transWH pairs. Together, the simulation and base substitution experiments predict that the coralyne-induced homo-(dA) duplex structure adopts the transWH geometry.

REBASE--a database for DNA restriction and modification: enzymes, genes and genomes

Roberts, R. J., Vincze, T., Posfai, J., Macelis, D. — Wed, 21 Oct 2009 07:49:02 PDT

REBASE is a comprehensive database of information about restriction enzymes, DNA methyltransferases and related proteins involved in the biological process of restriction–modification (R–M). It contains fully referenced information about recognition and cleavage sites, isoschizomers, neoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. Experimentally characterized homing endonucleases are also included. The fastest growing segment of REBASE contains the putative R–M systems found in the sequence databases. Comprehensive descriptions of the R–M content of all fully sequenced genomes are available including summary schematics. The contents of REBASE may be browsed from the web (http://rebase.neb.com) and selected compilations can be downloaded by ftp (ftp.neb.com). Additionally, monthly updates can be requested via email.

Selection of hyperfunctional siRNAs with improved potency and specificity

Wang, X., Wang, X., Varma, R. K., Beauchamp, L., Magdaleno, S., Sendera, T. J. — Wed, 21 Oct 2009 07:48:57 PDT

One critical step in RNA interference (RNAi) experiments is to design small interfering RNAs (siRNAs) that can greatly reduce the expression of the target transcripts, but not of other unintended targets. Although various statistical and computational approaches have been attempted, this remains a challenge facing RNAi researchers. Here, we present a new experimentally validated method for siRNA design. By analyzing public siRNA data and focusing on hyperfunctional siRNAs, we identified a set of sequence features as potency selection criteria to build an siRNA design algorithm with support vector machines. Additional bioinformatics filters were also included in the algorithm to increase RNAi specificity by reducing potential sequence cross-hybridization or microRNA-like effects. Independent validation experiments were performed, which indicated that the newly designed siRNAs have significantly improved performance, and worked effectively even at low concentrations. Furthermore, our cell-based studies demonstrated that the siRNA off-target effects were significantly reduced when the siRNAs were delivered into cells at the 3 nM concentration compared to 30 nM. Thus, the capability of our new design program to select highly potent siRNAs also renders increased RNAi specificity because these siRNAs can be used at a much lower concentration. The siRNA design web server is available at http://www5.appliedbiosystems.com/tools/siDesign/.

Novel recognition motifs and biological functions of the RNA-binding protein HuD revealed by genome-wide identification of its targets

Bolognani, F., Contente-Cuomo, T., Perrone-Bizzozero, N. I. — Wed, 21 Oct 2009 07:48:52 PDT

HuD is a neuronal ELAV-like RNA-binding protein (RBP) involved in nervous system development, regeneration, and learning and memory. This protein stabilizes mRNAs by binding to AU-rich instability elements (AREs) in their 3' unstranslated regions (3' UTR). To isolate its in vivo targets, messenger ribonucleoprotein (mRNP) complexes containing HuD were first immunoprecipitated from brain extracts and directly bound mRNAs identified by subsequent GST-HuD pull downs and microarray assays. Using the 3' UTR sequences of the most enriched targets and the known sequence restrictions of the HuD ARE-binding site, we discovered three novel recognition motifs. Motifs 2 and 3 are U-rich whereas motif 1 is C-rich. In vitro binding assays indicated that HuD binds motif 3 with the highest affinity, followed by motifs 2 and 1, with less affinity. These motifs were found to be over-represented in brain mRNAs that are upregulated in HuD overexpressor mice, supporting the biological function of these sequences. Gene ontology analyses revealed that HuD targets are enriched in signaling pathways involved in neuronal differentiation and that many of these mRNAs encode other RBPs, translation factors and actin-binding proteins. These findings provide further insights into the post-transcriptional mechanisms by which HuD promotes neural development and synaptic plasticity.

Transcriptome profiling defines a novel regulon modulated by the LysR-type transcriptional regulator MexT in Pseudomonas aeruginosa

Tian, Z.-X., Fargier, E., Mac Aogain, M., Adams, C., Wang, Y.-P., O'Gara, F. — Wed, 21 Oct 2009 07:48:44 PDT

The LysR-family regulator MexT modulates the expression of the MexEF-OprN efflux system in the human pathogen Pseudomonas aeruginosa. Recently, we demonstrated that MexT regulates certain virulence phenotypes, including the type-three secretion system and early attachment independent of its role in regulating MexEF-OprN. In this study, transcriptome profiling was utilized to investigate the global nature of MexT regulation in P. aeruginosa PAO1 and an isogenic mexEF mutant. Twelve genes of unknown function were highly induced by overexpressing MexT independent of MexEF-OprN. A well-conserved DNA motif was identified in the upstream regulatory region of nine of these genes and upstream of mexE. Reporter fusion analysis demonstrated that the expression of the genes was significantly induced by MexT in P. aeruginosa and a heterogenous Escherichia coli strain and that the conserved sequence was required for this induction. The conserved DNA motif was further characterized as the MexT binding site by site-directed mutagenesis and electrophoretic mobility shift assays. Genes containing this conserved regulatory sequence were identified across other Pseudomonas species, and their expression was activated by MexT. Thus, a novel regulon directly modulated by MexT, that includes but is independent of mexEF-oprN, has been identified.

5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy

Puffer, B., Kreutz, C., Rieder, U., Ebert, M.-O., Konrat, R., Micura, R. — Tue, 20 Oct 2009 07:03:51 PDT

¹⁹F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D ¹⁹F and ¹H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis.

Inactive X chromosome-specific histone H3 modifications and CpG hypomethylation flank a chromatin boundary between an X-inactivated and an escape gene

Goto, Y., Kimura, H. — Tue, 20 Oct 2009 07:03:45 PDT

In mammals, the dosage compensation of sex chromosomes between males and females is achieved by transcriptional inactivation of one of the two X chromosomes in females. However, a number of genes escape X-inactivation in humans. It remains poorly understood how the transcriptional activity of these ‘escape genes’ is maintained despite the chromosome-wide heterochromatin formation. To address this question, we analyzed a putative chromatin boundary between the inactivated RBM10 and an escape gene, UBA1/UBE1. Chromatin immunoprecipitation revealed that trimethylated histone H3 lysine 9 and H4 lysine 20 were enriched in the last exon through the proximal downstream region of RBM10, but were remarkably diminished at ~2 kb upstream of the UBA1 transcription start site. Whereas RNA polymerase II was not loaded onto the intergenic region, CTCF (CCCTC binding factor) was enriched around the boundary, where some CpG sites were hypomethylated specifically on inactive X. These findings suggest that local DNA hypomethylation and CTCF binding are involved in the formation of a chromatin boundary, which protects the UBA1 escape gene against the chromosome-wide transcriptional silencing.

Promoter activity of the sea urchin (Paracentrotus lividus) nucleosomal H3 and H2A and linker H1 {alpha}-histone genes is modulated by enhancer and chromatin insulator

Cavalieri, V., Melfi, R., Spinelli, G. — Tue, 20 Oct 2009 07:03:38 PDT

Core promoters and chromatin insulators are key regulatory elements that may direct a transcriptional enhancer to prefer a specific promoter in complex genetic loci. Enhancer and insulator flank the sea urchin (Paracentrotus lividus) -histone H2A transcription unit in a tandem repeated cluster containing the five histone genes. This article deals with the specificity of interaction between the H2A enhancer-bound MBF-1 activator and histone gene promoters, and with the mechanism that leads the H1 transcripts to peak at about one-third of the value for nucleosomal H3 and H2A mRNAs. To this end, in vivo competition assays of enhancer and insulator functions were performed. Our evidence suggests that the MBF-1 transcription factor participates also in the expression of the H3 gene and that the sns5 insulator buffers the downstream H1 promoter from the H2A enhancer. Altogether, these results provide a clear demonstration of the enhancer-blocking function of a chromatin insulator in a natural gene context. In addition, they suggest that both the H2A enhancer and the sns5 insulator may account for the diverse accumulation of the linker H1 versus the core nucleosomal histones during early development of the sea urchin embryo.

Expression profiling of Drosophila mitochondrial genes via deep mRNA sequencing

Torres, T. T., Dolezal, M., Schlotterer, C., Ottenwalder, B. — Tue, 20 Oct 2009 07:03:33 PDT

Mitochondria play an essential role in several cellular processes. Nevertheless, very little is known about patterns of gene expression of genes encoded by the mitochondrial DNA (mtDNA). In this study, we used next-generation sequencing (NGS) for transcription profiling of genes encoded in the mitochondrial genome of Drosophila melanogaster and D. pseudoobscura. The analysis of males and females in both species indicated that the expression pattern was conserved between the two species, but differed significantly between both sexes. Interestingly, mRNA levels were not only different among genes encoded by separate transcription units, but also showed significant differences among genes located in the same transcription unit. Hence, mRNA abundance of genes encoded by mtDNA seems to be heavily modulated by post-transcriptional regulation. Finally, we also identified several transcripts with a noncanonical structure, suggesting that processing of mitochondrial transcripts may be more complex than previously assumed.

TriTrypDB: a functional genomic resource for the Trypanosomatidae

Tue, 20 Oct 2009 07:03:30 PDT

TriTrypDB (http://tritrypdb.org) is an integrated database providing access to genome-scale datasets for kinetoplastid parasites, and supporting a variety of complex queries driven by research and development needs. TriTrypDB is a collaborative project, utilizing the GUS/WDK computational infrastructure developed by the Eukaryotic Pathogen Bioinformatics Resource Center (EuPathDB.org) to integrate genome annotation and analyses from GeneDB and elsewhere with a wide variety of functional genomics datasets made available by members of the global research community, often pre-publication. Currently, TriTrypDB integrates datasets from Leishmania braziliensis, L. infantum, L. major, L. tarentolae, Trypanosoma brucei and T. cruzi. Users may examine individual genes or chromosomal spans in their genomic context, including syntenic alignments with other kinetoplastid organisms. Data within TriTrypDB can be interrogated utilizing a sophisticated search strategy system that enables a user to construct complex queries combining multiple data types. All search strategies are stored, allowing future access and integrated searches. ‘User Comments’ may be added to any gene page, enhancing available annotation; such comments become immediately searchable via the text search, and are forwarded to curators for incorporation into the reference annotation when appropriate.

Pathema: a clade-specific bioinformatics resource center for pathogen research

Tue, 20 Oct 2009 07:03:26 PDT

Pathema (http://pathema.jcvi.org) is one of the eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infectious Disease (NIAID) designed to serve as a core resource for the bio-defense and infectious disease research community. Pathema strives to support basic research and accelerate scientific progress for understanding, detecting, diagnosing and treating an established set of six target NIAID Category A–C pathogens: Category A priority pathogens; Bacillus anthracis and Clostridium botulinum, and Category B priority pathogens; Burkholderia mallei, Burkholderia pseudomallei, Clostridium perfringens and Entamoeba histolytica. Each target pathogen is represented in one of four distinct clade-specific Pathema web resources and underlying databases developed to target the specific data and analysis needs of each scientific community. All publicly available complete genome projects of phylogenetically related organisms are also represented, providing a comprehensive collection of organisms for comparative analyses. Pathema facilitates the scientific exploration of genomic and related data through its integration with web-based analysis tools, customized to obtain, display, and compute results relevant to ongoing pathogen research. Pathema serves the bio-defense and infectious disease research community by disseminating data resulting from pathogen genome sequencing projects and providing access to the results of inter-genomic comparisons for these organisms.

The Universal Protein Resource (UniProt) in 2010

The UniProt Consortium — Tue, 20 Oct 2009 07:03:23 PDT

The primary mission of UniProt is to support biological research by maintaining a stable, comprehensive, fully classified, richly and accurately annotated protein sequence knowledgebase, with extensive cross-references and querying interfaces freely accessible to the scientific community. UniProt is produced by the UniProt Consortium which consists of groups from the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is comprised of four major components, each optimized for different uses: the UniProt Archive, the UniProt Knowledgebase, the UniProt Reference Clusters and the UniProt Metagenomic and Environmental Sequence Database. UniProt is updated and distributed every 3 weeks and can be accessed online for searches or download at http://www.uniprot.org.

Inferred Biomolecular Interaction Server--a web server to analyze and predict protein interacting partners and binding sites

Tue, 20 Oct 2009 07:03:21 PDT

IBIS is the NCBI Inferred Biomolecular Interaction Server. This server organizes, analyzes and predicts interaction partners and locations of binding sites in proteins. IBIS provides annotations for different types of binding partners (protein, chemical, nucleic acid and peptides), and facilitates the mapping of a comprehensive biomolecular interaction network for a given protein query. IBIS reports interactions observed in experimentally determined structural complexes of a given protein, and at the same time IBIS infers binding sites/interacting partners by inspecting protein complexes formed by homologous proteins. Similar binding sites are clustered together based on their sequence and structure conservation. To emphasize biologically relevant binding sites, several algorithms are used for verification in terms of evolutionary conservation, biological importance of binding partners, size and stability of interfaces, as well as evidence from the published literature. IBIS is updated regularly and is freely accessible via http://www.ncbi.nlm.nih.gov/Structure/ibis/ibis.html.

Sequence-non-specific effects of RNA interference triggers and microRNA regulators

Olejniczak, M., Galka, P., Krzyzosiak, W. J. — Tue, 20 Oct 2009 07:03:18 PDT

RNA reagents of diverse lengths and structures, unmodified or containing various chemical modifications are powerful tools of RNA interference and microRNA technologies. These reagents which are either delivered to cells using appropriate carriers or are expressed in cells from suitable vectors often cause unintended sequence-non-specific immune responses besides triggering intended sequence-specific silencing effects. This article reviews the present state of knowledge regarding the cellular sensors of foreign RNA, the signaling pathways these sensors mobilize and shows which specific features of the RNA reagents set the responsive systems on alert. The representative examples of toxic effects caused in the investigated cell lines and tissues by the RNAs of specific types and structures are collected and may be instructive for further studies of sequence-non-specific responses to foreign RNA in human cells.

Bind-n-Seq: high-throughput analysis of in vitro protein-DNA interactions using massively parallel sequencing

Zykovich, A., Korf, I., Segal, D. J. — Tue, 20 Oct 2009 07:03:16 PDT

Transcription factor–DNA interactions are some of the most important processes in biology because they directly control hereditary information. The targets of most transcription factor are unknown. In this report, we introduce Bind-n-Seq, a new high-throughput method for analyzing protein–DNA interactions in vitro, with several advantages over current methods. The procedure has three steps (i) binding proteins to randomized oligonucleotide DNA targets, (ii) sequencing the bound oligonucleotide with massively parallel technology and (iii) finding motifs among the sequences. De novo binding motifs determined by this method for the DNA-binding domains of two well-characterized zinc-finger proteins were similar to those described previously. Furthermore, calculations of the relative affinity of the proteins for specific DNA sequences correlated significantly with previous studies (R²= 0.9). These results present Bind-n-Seq as a highly rapid and parallel method for determining in vitro binding sites and relative affinities.

Modulation of the DNA-binding activity of Saccharomyces cerevisiae MSH2-MSH6 complex by the high-mobility group protein NHP6A, in vitro

Labazi, M., Jaafar, L., Flores-Rozas, H. — Tue, 20 Oct 2009 07:03:12 PDT

DNA mismatch repair corrects mispaired bases and small insertions/deletions in DNA. In eukaryotes, the mismatch repair complex MSH2–MSH6 binds to mispairs with only slightly higher affinity than to fully paired DNA in vitro. Recently, the high-mobility group box1 protein, (HMGB1), has been shown to stimulate the mismatch repair reaction in vitro. In yeast, the closest homologs of HMGB1 are NHP6A and NHP6B. These proteins have been shown to be required for genome stability maintenance and mutagenesis control. In this work, we show that MSH2–MSH6 and NHP6A modulate their binding to DNA in vitro. Binding of the yeast MSH2–MSH6 to homoduplex regions of DNA significantly stimulates the loading of NHP6A. Upon binding of NHP6A to DNA, MSH2–MSH6 is excluded from binding unless a mismatch is present. A DNA binding-impaired MSH2–MSH6F337A significantly reduced the loading of NHP6A to DNA, suggesting that MSH2–MSH6 binding is a requisite for NHP6A loading. MSH2–MSH6 and NHP6A form a stable complex, which is responsive to ATP on mismatched substrates. These results suggest that MSH2–MSH6 binding to homoduplex regions of DNA recruits NHP6A, which then prevents further binding of MSH2–MSH6 to these sites unless a mismatch is present.

Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human

Kuchta, K., Knizewski, L., Wyrwicz, L. S., Rychlewski, L., Ginalski, K. — Thu, 15 Oct 2009 04:36:48 PDT

This article presents a comprehensive review of large and highly diverse superfamily of nucleotidyltransferase fold proteins by providing a global picture about their evolutionary history, sequence-structure diversity and fulfilled functional roles. Using top-of-the-line homology detection method combined with transitive searches and fold recognition, we revised the realm of these superfamily in numerous databases of catalogued protein families and structures, and identified 10 new families of nucleotidyltransferase fold. These families include hundreds of previously uncharacterized and various poorly annotated proteins such as Fukutin/LICD, NFAT, FAM46, Mab-21 and NRAP. Some of these proteins seem to play novel important roles, not observed before for this superfamily, such as regulation of gene expression or choline incorporation into cell membrane. Importantly, within newly detected families we identified 25 novel superfamily members in human genome. Among these newly assigned members are proteins known to be involved in congenital muscular dystrophy, neurological diseases and retinal pigmentosa what sheds some new light on the molecular background of these genetic disorders. Twelve of new human nucleotidyltransferase fold proteins belong to Mab-21 family known to be involved in organogenesis and development. The determination of specific biological functions of these newly detected proteins remains a challenging task.

Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair

Wed, 14 Oct 2009 05:33:46 PDT

The Comet assay (CA) is a sensitive/simple measure of genotoxicity. However, many features of CA contribute variability. To minimize these, we have introduced internal standard materials consisting of ‘reference’ cells which have their DNA substituted with BrdU. Using a fluorescent anti-BrdU antibody, plus an additional barrier filter, comets derived from these cells could be readily distinguished from the ‘test’-cell comets, present in the same gel. In experiments to evaluate the reference cell comets as external and internal standards, the reference and test cells were present in separate gels on the same slide or mixed together in the same gel, respectively, before their co-exposure to X-irradiation. Using the reference cell comets as internal standards led to substantial reductions in the coefficient of variation (CoV) for intra- and inter-experimental measures of comet formation and DNA damage repair; only minor reductions in CoV were noted when the reference and test cell comets were in separate gels. These studies indicate that differences between individual gels appreciably contribute to CA variation. Further studies using the reference cells as internal standards allowed greater significance to be obtained between groups of replicate samples. Ultimately, we anticipate that development will deliver robust quality assurance materials for CA.

The Crc global regulator binds to an unpaired A-rich motif at the Pseudomonas putida alkS mRNA coding sequence and inhibits translation initiation

Moreno, R., Marzi, S., Romby, P., Rojo, F. — Tue, 13 Oct 2009 07:42:53 PDT

Crc is a key global translational regulator in Pseudomonads that orchestrates the hierarchy of induction of several catabolic pathways for amino acids, sugars, hydrocarbons or aromatic compounds. In the presence of amino acids, which are preferred carbon sources, Crc inhibits translation of the Pseudomonas putida alkS and benR mRNAs, which code for transcriptional regulators of genes required to assimilate alkanes (hydrocarbons) and benzoate (an aromatic compound), respectively. Crc binds to the 5'-end of these mRNAs, but the sequence and/or structure recognized, and the way in which it inhibits translation, were unknown. We have determined the secondary structure of the alkS mRNA 5'-end through its sensitivity to several ribonucleases and chemical reagents. Footprinting and band-shift assays using variant alkS mRNAs have shown that Crc specifically binds to a short unpaired A-rich sequence located adjacent to the alkS AUG start codon. This interaction is stable enough to prevent formation of the translational initiation complex. A similar Crc-binding site was localized at benR mRNA, upstream of the Shine–Dalgarno sequence. This allowed predicting binding sites at other Crc-regulated genes, deriving a consensus sequence that will help to validate new Crc targets and to discriminate between direct and indirect effects of this regulator.

Local false discovery rate facilitates comparison of different microarray experiments

Hong, W.-J., Tibshirani, R., Chu, G. — Tue, 13 Oct 2009 07:42:46 PDT

The local false discovery rate (LFDR) estimates the probability of falsely identifying specific genes with changes in expression. In computer simulations, LFDR <10% successfully identified genes with changes in expression, while LFDR >90% identified genes without changes. We used LFDR to compare different microarray experiments quantitatively: (i) Venn diagrams of genes with and without changes in expression, (ii) scatter plots of the genes, (iii) correlation coefficients in the scatter plots and (iv) distributions of gene function. To illustrate, we compared three methods for pre-processing microarray data. Correlations between methods were high (r = 0.84–0.92). However, responses were often different in magnitude, and sometimes discordant, even though the methods used the same raw data. LFDR complements functional assessments like gene set enrichment analysis. To illustrate, we compared responses to ultraviolet radiation (UV), ionizing radiation (IR) and tobacco smoke. Compared to unresponsive genes, genes responsive to both UV and IR were enriched for cell cycle, mitosis, and DNA repair functions. Genes responsive to UV but not IR were depleted for cell adhesion functions. Genes responsive to tobacco smoke were enriched for detoxification functions. Thus, LFDR reveals differences and similarities among experiments.

Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines

Tue, 13 Oct 2009 07:42:41 PDT

The X chromosome-linked inhibitor of apoptosis protein (XIAP) is the most potent intrinsic caspase inhibitor and plays an important role in the maintenance of intestinal epithelial integrity. The RNA binding protein, HuR, regulates the stability and translation of many target transcripts. Here, we report that HuR associated with both the 3'-untranslated region and coding sequence of the mRNA encoding XIAP, stabilized the XIAP transcript and elevated its expression in intestinal epithelial cells. Ectopic HuR overexpression or elevated cytoplasmic levels of endogenous HuR by decreasing cellular polyamines increased [HuR/XIAP mRNA] complexes, in turn promoting XIAP mRNA stability and increasing XIAP protein abundance. Conversely, HuR silencing in normal and polyamine-deficient cells rendered the XIAP mRNA unstable, thus reducing the steady state levels of XIAP. Inhibition of XIAP expression by XIAP silencing or by HuR silencing reversed the resistance of polyamine-deficient cells to apoptosis. Our findings demonstrate that HuR regulates XIAP expression by stabilizing its mRNA and implicates HuR-mediated XIAP in the control of intestinal epithelial apoptosis.

Molecular interactions between HNF4a, FOXA2 and GABP identified at regulatory DNA elements through ChIP-sequencing

Mon, 12 Oct 2009 01:48:21 PDT

Gene expression is regulated by combinations of transcription factors, which can be mapped to regulatory elements on a genome-wide scale using ChIP experiments. In a previous ChIP-chip study of USF1 and USF2 we found evidence also of binding of GABP, FOXA2 and HNF4a within the enriched regions. Here, we have applied ChIP-seq for these transcription factors and identified 3064 peaks of enrichment for GABP, 7266 for FOXA2 and 18783 for HNF4a. Distal elements with USF2 signal was frequently bound also by HNF4a and FOXA2. GABP peaks were found at transcription start sites, whereas 94% of FOXA2 and 90% of HNF4a peaks were located at other positions. We developed a method to accurately define TFBS within peaks, and found the predicted sites to have an elevated conservation level compared to peak centers; however the majority of bindings were not evolutionary conserved. An interaction between HNF4a and GABP was seen at TSS, with one-third of the HNF4a positive promoters being bound also by GABP, and this interaction was verified by co-immunoprecipitations.

More than a decade of developmental gene expression atlases: where are we now?

de Boer, B. A., Ruijter, J. M., Voorbraak, F. P. J. M., Moorman, A. F. M. — Mon, 12 Oct 2009 01:48:05 PDT

To unravel regulatory networks of genes functioning during embryonic development, information on in situ gene expression is required. Enormous amounts of such data are available in literature, where each paper reports on a limited number of genes and developmental stages. The best way to make these data accessible is via spatio-temporal gene expression atlases. Eleven atlases, describing developing vertebrates and covering at least 100 genes, were reviewed. This review focuses on: (i) the used anatomical framework, (ii) the handling of input data and (iii) the retrieval of information. Our aim is to provide insights into both the possibilities of the atlases, as well as to describe what more than a decade of developmental gene expression atlases can teach us about the requirements of the design of the ‘ideal atlas’. This review shows that most ingredients needed to develop the ideal atlas are already applied to some extent in at least one of the discussed atlases. A review of these atlases shows that the ideal atlas should be based on a spatial framework, i.e. a series of 3D reference models, which is anatomically annotated using an ontology with sufficient resolution, both for relations as well as for anatomical terms.

Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

Macickova-Cahova, H., Hocek, M. — Fri, 09 Oct 2009 08:34:36 PDT

A set of 6 base-modified 2'-deoxyadenosine derivatives was incorporated to diverse DNA sequences by primer extension using Vent (exo-) polymerase and the influence of the modification on cleavage by diverse restriction endonucleases was studied. While 8-substituted (Br or methyl) adenine derivatives were well tolerated by the restriction enzymes and the corresponding sequences were cleaved, the presence of 7-substituted 7-deazaadenine in the recognition sequence resulted in blocking of cleavage by some enzymes depending on the nature and size of the 7-substituent. All sequences with modifications outside of the recognition sequence were perfectly cleaved by all the restriction enzymes. The results are useful both for protection of some sequences from cleavage and for manipulation of functionalized DNA by restriction cleavage.

Stabilization of G-quadruplex in the BCL2 promoter region in double-stranded DNA by invading short PNAs

Fri, 09 Oct 2009 08:34:31 PDT

Numerous regulatory genes have G-rich regions that can potentially form quadruplex structures, possibly playing a role in transcription regulation. We studied a G-rich sequence in the BCL2 gene 176-bp upstream of the P1 promoter for G-quadruplex formation. Using circular dichroism (CD), thermal denaturation and dimethyl sulfate (DMS) footprinting, we found that a single-stranded oligonucleotide with the sequence of the BCL2 G-rich region forms a potassium-stabilized G-quadruplex. To study G-quadruplex formation in double-stranded DNA, the G-rich sequence of the BCL2 gene was inserted into plasmid DNA. We found that a G-quadruplex did not form in the insert at physiological conditions. To induce G-quadruplex formation, we used short peptide nucleic acids (PNAs) that bind to the complementary C-rich strand. We examined both short duplex-forming PNAs, complementary to the central part of the BCL2 gene, and triplex-forming bis-PNAs, complementary to sequences adjacent to the G-rich BCL2 region. Using a DMS protection assay, we demonstrated G-quadruplex formation within the G-rich sequence from the promoter region of the human BCL2 gene in plasmid DNA. Our results show that molecules binding the complementary C-strand facilitate G-quadruplex formation and introduce a new mode of PNA-mediated sequence-specific targeting.

SelTarbase, a database of human mononucleotide-microsatellite mutations and their potential impact to tumorigenesis and immunology

Fri, 09 Oct 2009 08:34:20 PDT

About 15% of human colorectal cancers and, at varying degrees, other tumor entities as well as nearly all tumors related to Lynch syndrome are hallmarked by microsatellite instability (MSI) as a result of a defective mismatch repair system. The functional impact of resulting mutations depends on their genomic localization. Alterations within coding mononucleotide repeat tracts (MNRs) can lead to protein truncation and formation of neopeptides, whereas alterations within untranslated MNRs can alter transcription level or transcript stability. These mutations may provide selective advantage or disadvantage to affected cells. They may further concern the biology of microsatellite unstable cells, e.g. by generating immunogenic peptides induced by frameshifts mutations. The Selective Targets database (http://www.seltarbase.org) is a curated database of a growing number of public MNR mutation data in microsatellite unstable human tumors. Regression calculations for various MSI–H tumor entities indicating statistically deviant mutation frequencies predict TGFBR2, BAX, ACVR2A and others that are shown or highly suspected to be involved in MSI tumorigenesis. Many useful tools for further analyzing genomic DNA, derived wild-type and mutated cDNAs and peptides are integrated. A comprehensive database of all human coding, untranslated, non-coding RNA- and intronic MNRs (MNR_ensembl) is also included. Herewith, SelTarbase presents as a plenty instrument for MSI-carcinogenesis-related research, diagnostics and therapy.

TMEM8 - a non-globin gene entrapped in the globin web

Fri, 09 Oct 2009 08:34:13 PDT

For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of -globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the -globin cluster due to inversion of an ~170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the -globin genes. Surprisingly, TMEM8 is not simply recruited to the -globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs.

Sub-cellular trafficking and functionality of 2'-O-methyl and 2'-O-methyl-phosphorothioate molecular beacons

Chen, A. K., Behlke, M. A., Tsourkas, A. — Fri, 09 Oct 2009 08:34:10 PDT

Molecular beacons (MBs) have shown great potential for the imaging of RNAs within single living cells; however, the ability to perform accurate measurements of RNA expression can be hampered by false-positives resulting from nonspecific interactions and/or nuclease degradation. These false-positives could potentially be avoided by introducing chemically modified oligonucleotides into the MB design. In this study, fluorescence microscopy experiments were performed to elucidate the subcellular trafficking, false-positive signal generation, and functionality of 2'-O-methyl (2Me) and 2'-O-methyl-phosphorothioate (2MePS) MBs. The 2Me MBs exhibited rapid nuclear sequestration and a gradual increase in fluorescence over time, with nearly 50% of the MBs being opened nonspecifically within 24 h. In contrast, the 2MePS MBs elicited an instantaneous increase in false-positives, corresponding to ~5–10% of the MBs being open, but little increase was observed over the next 24 h. Moreover, trafficking to the nucleus was slower. After 24 h, both MBs were localized in the nucleus and lysosomal compartments, but only the 2MePS MBs were still functional. When the MBs were retained in the cytoplasm, via conjugation to NeutrAvidin, a significant reduction in false-positives and improvement in functionality was observed. Overall, these results have significant implications for the design and applications of MBs for intracellular RNA measurement.

DNaseI hypersensitivity at gene-poor, FSH dystrophy-linked 4q35.2

Fri, 09 Oct 2009 08:34:06 PDT

A subtelomeric region, 4q35.2, is implicated in facioscapulohumeral muscular dystrophy (FSHD), a dominant disease thought to involve local pathogenic changes in chromatin. FSHD patients have too few copies of a tandem 3.3-kb repeat (D4Z4) at 4q35.2. No phenotype is associated with having few copies of an almost identical repeat at 10q26.3. Standard expression analyses have not given definitive answers as to the genes involved. To investigate the pathogenic effects of short D4Z4 arrays on gene expression in the very gene-poor 4q35.2 and to find chromatin landmarks there for transcription control, unannotated genes and chromatin structure, we mapped DNaseI-hypersensitive (DH) sites in FSHD and control myoblasts. Using custom tiling arrays (DNase-chip), we found unexpectedly many DH sites in the two large gene deserts in this 4-Mb region. One site was seen preferentially in FSHD myoblasts. Several others were mapped >0.7 Mb from genes known to be active in the muscle lineage and were also observed in cultured fibroblasts, but not in lymphoid, myeloid or hepatic cells. Their selective occurrence in cells derived from mesoderm suggests functionality. Our findings indicate that the gene desert regions of 4q35.2 may have functional significance, possibly also to FSHD, despite their paucity of known genes.

AntigenDB: an immunoinformatics database of pathogen antigens

Ansari, H. R., Flower, D. R., Raghava, G. P. S. — Fri, 09 Oct 2009 08:34:03 PDT

The continuing threat of infectious disease and future pandemics, coupled to the continuous increase of drug-resistant pathogens, makes the discovery of new and better vaccines imperative. For effective vaccine development, antigen discovery and validation is a prerequisite. The compilation of information concerning pathogens, virulence factors and antigenic epitopes has resulted in many useful databases. However, most such immunological databases focus almost exclusively on antigens where epitopes are known and ignore those for which epitope information was unavailable. We have compiled more than 500 antigens into the AntigenDB database, making use of the literature and other immunological resources. These antigens come from 44 important pathogenic species. In AntigenDB, a database entry contains information regarding the sequence, structure, origin, etc. of an antigen with additional information such as B and T-cell epitopes, MHC binding, function, gene-expression and post translational modifications, where available. AntigenDB also provides links to major internal and external databases. We shall update AntigenDB on a rolling basis, regularly adding antigens from other organisms and extra data analysis tools. AntigenDB is available freely at http://www.imtech.res.in/raghava/antigendb and its mirror site http://www.bic.uams.edu/raghava/antigendb.

High DNA melting temperature predicts transcription start site location in human and mouse

Dineen, D. G., Wilm, A., Cunningham, P., Higgins, D. G. — Fri, 09 Oct 2009 08:34:00 PDT

The accurate computational prediction of transcription start sites (TSS) in vertebrate genomes is a difficult problem. The physicochemical properties of DNA can be computed in various ways and a many combinations of DNA features have been tested in the past for use as predictors of transcription. We looked in detail at melting temperature, which measures the temperature, at which two strands of DNA separate, considering the cooperative nature of this process. We find that peaks in melting temperature correspond closely to experimentally determined transcription start sites in human and mouse chromosomes. Using melting temperature alone, and with simple thresholding, we can predict TSS with accuracy that is competitive with the most accurate state-of-the-art TSS prediction methods. Accuracy is measured using both experimentally and manually determined TSS. The method works especially well with CpG island containing promoters, but also works when CpG islands are absent. This result is clear evidence of the important role of the physical properties of DNA in the process of transcription. It also points to the importance for TSS prediction methods to include melting temperature as prior information.

Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy

Fri, 09 Oct 2009 08:33:56 PDT

Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2'-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains—the A-site finger and Helix 69 (six and five modifications). Results show that a number of modifications influence accuracy with effects ranging from 0.3- to 2.4-fold of wild-type activity. Blocking subsets of modifications, especially from the decoding region, impairs stop codon termination and reading frame maintenance. Unexpectedly, several Helix 69 mutants possess ribosomes with increased fidelity. Consistent with strong positional and synergistic effects is the finding that single deletions can have a more pronounced phenotype than multiple deficiencies in the same region. Altogether, the results demonstrate that rRNA modifications have significant roles in translation accuracy.

Dynamic Proteomics: a database for dynamics and localizations of endogenous fluorescently-tagged proteins in living human cells

Fri, 09 Oct 2009 08:33:53 PDT

Recent advances allow tracking the levels and locations of a thousand proteins in individual living human cells over time using a library of annotated reporter cell clones (LARC). This library was created by Cohen et al. to study the proteome dynamics of a human lung carcinoma cell-line treated with an anti-cancer drug. Here, we report the Dynamic Proteomics database for the proteins studied by Cohen et al. Each cell-line clone in LARC has a protein tagged with yellow fluorescent protein, expressed from its endogenous chromosomal location, under its natural regulation. The Dynamic Proteomics interface facilitates searches for genes of interest, downloads of protein fluorescent movies and alignments of dynamics following drug addition. Each protein in the database is displayed with its annotation, cDNA sequence, fluorescent images and movies obtained by the time-lapse microscopy. The protein dynamics in the database represents a quantitative trace of the protein fluorescence levels in nucleus and cytoplasm produced by image analysis of movies over time. Furthermore, a sequence analysis provides a search and comparison of up to 50 input DNA sequences with all cDNAs in the library. The raw movies may be useful as a benchmark for developing image analysis tools for individual-cell dynamic-proteomics. The database is available at http://www.dynamicproteomics.net/.

BeetleBase in 2010: revisions to provide comprehensive genomic information for Tribolium castaneum

Fri, 09 Oct 2009 08:33:50 PDT

BeetleBase (http://www.beetlebase.org) has been updated to provide more comprehensive genomic information for the red flour beetle Tribolium castaneum. The database contains genomic sequence scaffolds mapped to 10 linkage groups (genome assembly release Tcas_3.0), genetic linkage maps, the official gene set, Reference Sequences from NCBI (RefSeq), predicted gene models, ESTs and whole-genome tiling array data representing several developmental stages. The database was reconstructed using the upgraded Generic Model Organism Database (GMOD) modules. The genomic data is stored in a PostgreSQL relatational database using the Chado schema and visualized as tracks in GBrowse. The updated genetic map is visualized using the comparative genetic map viewer CMAP. To enhance the database search capabilities, the BLAST and BLAT search tools have been integrated with the GMOD tools. BeetleBase serves as a long-term repository for Tribolium genomic data, and is compatible with other model organism databases.

Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs

Thu, 08 Oct 2009 08:31:09 PDT

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.

A simple method for directional transcriptome sequencing using Illumina technology

Thu, 08 Oct 2009 08:31:05 PDT

High-throughput sequencing of cDNA has been used to study eukaryotic transcription on a genome-wide scale to single base pair resolution. In order to compensate for the high ribonuclease activity in bacterial cells, we have devised an equivalent technique optimized for studying complete prokaryotic transcriptomes that minimizes the manipulation of the RNA sample. This new approach uses Illumina technology to sequence single-stranded (ss) cDNA, generating information on both the direction and level of transcription throughout the genome. The protocol, and associated data analysis programs, are freely available from http://www.sanger.ac.uk/Projects/Pathogens/Transcriptome/. We have successfully applied this method to the bacterial pathogens Salmonella bongori and Streptococcus pneumoniae and the yeast Schizosaccharomyces pombe. This method enables experimental validation of genetic features predicted in silico and allows the easy identification of novel transcripts throughout the genome. We also show that there is a high correlation between the level of gene expression calculated from ss-cDNA and double-stranded-cDNA sequencing, indicting that ss-cDNA sequencing is both robust and appropriate for use in quantitative studies of transcription. Hence, this simple method should prove a useful tool in aiding genome annotation and gene expression studies in both prokaryotes and eukaryotes.

A directed evolution design of a GCG-specific DNA hemimethylase

Gerasimaite, R., Vilkaitis, G., Klimasauskas, S. — Thu, 08 Oct 2009 09:37:24 PDT

DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases.

Interaction of the HIV-1 frameshift signal with the ribosome

Mazauric, M.-H., Seol, Y., Yoshizawa, S., Visscher, K., Fourmy, D. — Wed, 07 Oct 2009 06:39:43 PDT

Ribosomal frameshifting on viral RNAs relies on the mechanical properties of structural elements, often pseudoknots and more rarely stem-loops, that are unfolded by the ribosome during translation. In human immunodeficiency virus (HIV)-1 type B a long hairpin containing a three-nucleotide bulge is responsible for efficient frameshifting. This three-nucleotide bulge separates the hairpin in two domains: an unstable lower stem followed by a GC-rich upper stem. Toeprinting and chemical probing assays suggest that a hairpin-like structure is retained when ribosomes, initially bound at the slippery sequence, were allowed multiple EF-G catalyzed translocation cycles. However, while the upper stem remains intact the lower stem readily melts. After the first, and single step of translocation of deacylated tRNA to the 30 S P site, movement of the mRNA stem-loop in the 5' direction is halted, which is consistent with the notion that the downstream secondary structure resists unfolding. Mechanical stretching of the hairpin using optical tweezers only allows clear identification of unfolding of the upper stem at a force of 12.8 ± 1.0 pN. This suggests that the lower stem is unstable and may indeed readily unfold in the presence of a translocating ribosome.

New tools at the Candida Genome Database: biochemical pathways and full-text literature search

Tue, 06 Oct 2009 08:48:02 PDT

The Candida Genome Database (CGD, http://www.candidagenome.org/) provides online access to genomic sequence data and manually curated functional information about genes and proteins of the human pathogen Candida albicans. Herein, we describe two recently added features, Candida Biochemical Pathways and the Textpresso full-text literature search tool. The Biochemical Pathways tool provides visualization of metabolic pathways and analysis tools that facilitate interpretation of experimental data, including results of large-scale experiments, in the context of Candida metabolism. Textpresso for Candida allows searching through the full-text of Candida-specific literature, including clinical and epidemiological studies.

Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase {alpha}4 helix

Tue, 06 Oct 2009 08:47:59 PDT

HIV-1 integrase integrates retroviral DNA through 3'-processing and strand transfer reactions in the presence of a divalent cation (Mg²⁺ or Mn²⁺). The 4 helix exposed at the catalytic core surface is essential to the specific recognition of viral DNA. To define group determinants of recognition, we used a model composed of a peptide analogue of the 4 helix, oligonucleotides mimicking processed and unprocessed U5 LTR end and 5 mM Mg²⁺. Circular dichroism, fluorescence and NMR experiments confirmed the implication of the 4 helix polar/charged face in specific and non-specific bindings to LTR ends. The specific binding requires unprocessed LTR ends—i.e. an unaltered 3'-processing site CAGT3'—and is reinforced by Mg²⁺ (K_d decreases from 2 to 0.8 nM). The latter likely interacts with the ApG and GpT3' steps of the 3'-processing site. With deletion of GT3', only persists non-specific binding (K_d of 100 µM). Proton chemical shift deviations showed that specific binding need conserved amino acids in the 4 helix and conserved nucleotide bases and backbone groups at LTR ends. We suggest a conserved recognition mechanism based on both direct and indirect readout and which is subject to evolutionary pressure.

PMRD: plant microRNA database

Tue, 06 Oct 2009 08:47:55 PDT

MicroRNAs (miRNA) are ~21 nucleotide-long non-coding small RNAs, which function as post-transcriptional regulators in eukaryotes. miRNAs play essential roles in regulating plant growth and development. In recent years, research into the mechanism and consequences of miRNA action has made great progress. With whole genome sequence available in such plants as Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Glycine max, etc., it is desirable to develop a plant miRNA database through the integration of large amounts of information about publicly deposited miRNA data. The plant miRNA database (PMRD) integrates available plant miRNA data deposited in public databases, gleaned from the recent literature, and data generated in-house. This database contains sequence information, secondary structure, target genes, expression profiles and a genome browser. In total, there are 8433 miRNAs collected from 121 plant species in PMRD, including model plants and major crops such as Arabidopsis, rice, wheat, soybean, maize, sorghum, barley, etc. For Arabidopsis, rice, poplar, soybean, cotton, medicago and maize, we included the possible target genes for each miRNA with a predicted interaction site in the database. Furthermore, we provided miRNA expression profiles in the PMRD, including our local rice oxidative stress related microarray data (LC Sciences miRPlants_10.1) and the recently published microarray data for poplar, Arabidopsis, tomato, maize and rice. The PMRD database was constructed by open source technology utilizing a user-friendly web interface, and multiple search tools. The PMRD is freely available at http://bioinformatics.cau.edu.cn/PMRD. We expect PMRD to be a useful tool for scientists in the miRNA field in order to study the function of miRNAs and their target genes, especially in model plants and major crops.

Expanded RNA-binding activities of mammalian Argonaute 2

Tue, 06 Oct 2009 08:47:52 PDT

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.

DNA cleavage and methylation specificity of the single polypeptide restriction-modification enzyme LlaGI

Smith, R. M., Diffin, F. M., Savery, N. J., Josephsen, J., Szczelkun, M. D. — Tue, 06 Oct 2009 08:47:46 PDT

LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a -family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5'-CTnGAyG-3' (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5'-CrTCnAG-3' being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).

MEPE/OF45 protects cells from DNA damage induced killing via stabilizing CHK1

Liu, S., Wang, H., Wang, X., Lu, L., Gao, N., Rowe, P. S. N., Hu, B., Wang, Y. — Tue, 06 Oct 2009 08:47:41 PDT

Matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) was cloned in 2000 with functions related to bone metabolism. We identified MEPE/OF45 for the first time as a new co-factor of CHK1 in mammalian cells to protect cells from DNA damage induced killing. We demonstrate here that MEPE/OF45 directly interacts with CHK1. Knocking down MEPE/OF45 decreases CHK1 levels and sensitizes the cells to DNA damage inducers such as ionizing radiation (IR) or camptothicin (CPT)-induced killing. Over-expressing wild-type MEPE/OF45, but not the mutant MEPE/OF45 (depleted the key domain to interact with CHK1) increases CHK1 levels in the cells and increases the resistance of the cells to IR or CPT. MEPE/OF45, interacting with CHK1, increases CHK1 half-life and decreases CHK1 degradation through the ubiquitine-mediated pathway. In addition, the interaction of MEPE/OF45 with CHK1 decreases CHK1 levels in the ubiquitin E3 ligases (Cul1 and Cul4A) complex, which suggests that MEPE/OF45 competes with the ubiquitin E3 ligases binding to CHK1 and thus decreases CHK1 from ubiquitin-mediated proteolysis. These findings reveal an important role of MEPE/OF45 in protecting cells from DNA damage induced killing through stabilizing CHK1, which would provide MEPE/OF45 as a new target for sensitizing tumor cells to radiotherapy or chemotherapy.

PCR-free method detects high frequency of genomic instability in prostate cancer

Makridakis, N. M., Phipps, T., Srivastav, S., Reichardt, J. K. V. — Thu, 01 Oct 2009 05:11:33 PDT

Most studies of tumor instability are PCR-based. PCR-based methods may underestimate mutation frequencies of heterogeneous tumor genomes. Using a novel PCR-free random cloning/sequencing method, we analyzed 100 kb of total genomic DNA from blood lymphocytes, normal prostate and tumor prostate taken from six individuals. Variations were identified by comparison of the sequence of the cloned fragments with the nr-database in Genbank. After excluding known polymorphisms (by comparison to the NCBI dbSNP), we report a significant over-representation of variants in the tumors: 0.66 variations per kilobase of sequence, compared with the corresponding normal prostates (0.14 variations/kb) or blood (0.09 variations/kb). Extrapolating the observed difference between tumor and normal prostate DNA, we estimate 1.8 million somatic (de novo) alterations per tumor cell genome, a much higher frequency than previous measurements obtained by mostly PCR-based methods in other tumor types. Moreover, unlike the normal prostate and blood, most of the tumor variations occur in a specific motif (P = 0.046), suggesting common etiology. We further report high tumor cell-to-cell heterogeneity. These data have important implications for selecting appropriate technologies for cancer genome projects as well as for understanding prostate cancer progression.

SilkDB v2.0: a platform for silkworm (Bombyx mori ) genome biology

Wed, 30 Sep 2009 07:33:31 PDT

The SilkDB is an open-access database for genome biology of the silkworm (Bombyx mori). Since the draft sequence was completed and the SilkDB was first released 5 years ago, we have collaborated with other groups to make much remarkable progress on silkworm genome research, such as the completion of a new high-quality assembly of the silkworm genome sequence as well as the construction of a genome-wide microarray to survey gene expression profiles. To accommodate these new genomic data and house more comprehensive genomic information, we have reconstructed SilkDB database with new web interfaces. In the new version (v2.0) of SilkDB, we updated the genomic data, including genome assembly, gene annotation, chromosomal mapping, orthologous relationship and experiment data, such as microarray expression data, Expressed Sequence Tags (ESTs) and corresponding references. Several new tools, including SilkMap, Silkworm Chromosome Browser (SCB) and BmArray, are developed to access silkworm genomic data conveniently. SilkDB is publicly available at the new URL of http://www.silkdb.org.

An Mrr-family nuclease motif in the single polypeptide restriction-modification enzyme LlaGI

Smith, R. M., Josephsen, J., Szczelkun, M. D. — Wed, 30 Sep 2009 07:33:28 PDT

Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction–modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

Cuzic-Feltens, S., Weber, M. H. W., Hartmann, R. K. — Wed, 30 Sep 2009 07:33:25 PDT

We analyzed cleavage of precursor tRNAs with an LNA, 2'-OCH₃, 2'-H or 2'-F modification at the canonical (c₀) site by bacterial RNase P. We infer that the major function of the 2'-substituent at nt –1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c₀ site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3' -endo ribose and without the H-bond donor function of the 2'-substituent. LNA and 2'-OCH₃ suppressed processing at the major aberrant m_–₁ site; instead, the m₊₁ (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c₀ and m₊₁ sites had different pH profiles, with a higher Mg²⁺ requirement for c₀ versus m₊₁ cleavage. The strong catalytic defect for LNA and 2'-OCH₃ supports a model where the extra methylene (LNA) or methyl group (2'-OCH₃) causes a steric interference with a nearby bound catalytic Mg²⁺ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects.

Phage T4 mobE promotes trans homing of the defunct homing endonuclease I-TevIII

Wilson, G. W., Edgell, D. R. — Wed, 30 Sep 2009 07:33:22 PDT

Homing endonucleases are site-specific DNA endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the endonuclease, resulting in a unidirectional gene conversion event that mobilizes the homing endonuclease gene and flanking DNA. Here, we characterize phage T4-encoded mobE, a predicted free-standing HNH family homing endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct homing endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility.

Role and dynamics of the ribosomal protein P0 and its related trans-acting factor Mrt4 during ribosome assembly in Saccharomyces cerevisiae

Tue, 29 Sep 2009 08:13:34 PDT

Mrt4 is a nucleolar component of the ribosome assembly machinery that shares notable similarity and competes for binding to the 25S rRNA GAR domain with the ribosomal protein P0. Here, we show that loss of function of either P0 or Mrt4 results in a deficit in 60S subunits, which is apparently due to impaired rRNA processing of 27S precursors. Mrt4, which shuttles between the nucleus and the cytoplasm, defines medium pre-60S particles. In contrast, P0 is absent from medium but present in late/cytoplasmic pre-60S complexes. The absence of Mrt4 notably increased the amount of P0 in nuclear Nop7–TAP complexes and causes P0 assembly to medium pre-60S particles. Upon P0 depletion, Mrt4 is relocated to the cytoplasm within aberrant 60S subunits. We conclude that Mrt4 controls the position and timing of P0 assembly. In turn, P0 is required for the release of Mrt4 and exchanges with this factor at the cytoplasm. Our results also suggest other P0 assembly alternatives.

Overlapping promoter targeting by Elk-1 and other divergent ETS-domain transcription factor family members

Tue, 29 Sep 2009 08:13:30 PDT

ETS-domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targets for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targets we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.

The catalytic residues of Tn3 resolvase

Olorunniji, F. J., Stark, W. M. — Tue, 29 Sep 2009 08:13:25 PDT

To characterize the residues that participate in the catalysis of DNA cleavage and rejoining by the site-specific recombinase Tn3 resolvase, we mutated conserved polar or charged residues in the catalytic domain of an activated resolvase variant. We analysed the effects of mutations at 14 residues on proficiency in binding to the recombination site (‘site I’), formation of a synaptic complex between two site Is, DNA cleavage and recombination. Mutations of Y6, R8, S10, D36, R68 and R71 resulted in greatly reduced cleavage and recombination activity, suggesting crucial roles of these six residues in catalysis, whereas mutations of the other residues had less dramatic effects. No mutations strongly inhibited binding of resolvase to site I, but several caused conspicuous changes in the yield or stability of the synapse of two site Is observed by non-denaturing gel electrophoresis. The involvement of some residues in both synapsis and catalysis suggests that they contribute to a regulatory mechanism, in which engagement of catalytic residues with the substrate is coupled to correct assembly of the synapse.

Bicistronic DNA display for in vitro selection of Fab fragments

Sumida, T., Doi, N., Yanagawa, H. — Tue, 29 Sep 2009 08:13:21 PDT

In vitro display methods are superior tools for obtaining monoclonal antibodies. Although totally in vitro display methods, such as ribosome display and mRNA display, have the advantages of larger library sizes and quicker selection procedures compared with phage display, their applications have been limited to single-chain Fvs due to the requirement for linking of the mRNA and the nascent protein on the ribosome. Here we describe a different type of totally in vitro method, DNA display, that is applicable to heterodimeric Fab fragments: in vitro compartmentalization in water-in-oil emulsions allows the linking of an oligomeric protein and its encoding DNA with multiple ORFs. Since previously used emulsions impaired the synthesis of functional Fab fragments, we modified conditions for preparing emulsions, and identified conditions under which it was possible to enrich Fab fragments 10⁶-fold per three rounds of affinity selection. Furthermore, we confirmed that genes encoding stable Fab fragments could be selected from a Fab fragment library with a randomized hydrophobic core in the constant region by applying heat treatment as a selection pressure. Since this method has all advantages of both phage display and totally in vitro display, it represents a new option for many applications using display methods.

TransmiR: a transcription factor-microRNA regulation database

Wang, J., Lu, M., Qiu, C., Cui, Q. — Mon, 28 Sep 2009 05:41:55 PDT

MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are therefore important cellular components. As is true for protein-coding genes, the transcription of miRNAs is regulated by transcription factors (TFs), an important class of gene regulators that act at the transcriptional level. The correct regulation of miRNAs by TFs is critical, and increasing evidence indicates that aberrant regulation of miRNAs by TFs can cause phenotypic variations and diseases. Therefore, a TF–miRNA regulation database would be helpful for understanding the mechanisms by which TFs regulate miRNAs and understanding their contribution to diseases. In this study, we manually surveyed approximately 5000 reports in the literature and identified 243 TF–miRNA regulatory relationships, which were supported experimentally from 86 publications. We used these data to build a TF–miRNA regulatory database (TransmiR, http://cmbi.bjmu.edu.cn/transmir), which contains 82 TFs and 100 miRNAs with 243 regulatory pairs between TFs and miRNAs. In addition, we included references to the published literature (PubMed ID) information about the organism in which the relationship was found, whether the TFs and miRNAs are involved with tumors, miRNA function annotation and miRNA-associated disease annotation. TransmiR provides a user-friendly interface by which interested parties can easily retrieve TF–miRNA regulatory pairs by searching for either a miRNA or a TF.

A protein-protein interaction guided method for competitive transcription factor binding improves target predictions

Laurila, K., Yli-Harja, O., Lahdesmaki, H. — Mon, 28 Sep 2009 05:41:53 PDT

An important milestone in revealing cells' functions is to build a comprehensive understanding of transcriptional regulation processes. These processes are largely regulated by transcription factors (TFs) binding to DNA sites. Several TF binding site (TFBS) prediction methods have been developed, but they usually model binding of a single TF at a time albeit few methods for predicting binding of multiple TFs also exist. In this article, we propose a probabilistic model that predicts binding of several TFs simultaneously. Our method explicitly models the competitive binding between TFs and uses the prior knowledge of existing protein–protein interactions (PPIs), which mimics the situation in the nucleus. Modeling DNA binding for multiple TFs improves the accuracy of binding site prediction remarkably when compared with other programs and the cases where individual binding prediction results of separate TFs have been combined. The traditional TFBS prediction methods usually predict overwhelming number of false positives. This lack of specificity is overcome remarkably with our competitive binding prediction method. In addition, previously unpredictable binding sites can be detected with the help of PPIs. Source codes are available at http://www.cs.tut.fi/~harrila/.

Revisiting the planarity of nucleic acid bases: Pyramidilization at glycosidic nitrogen in purine bases is modulated by orientation of glycosidic torsion

Mon, 28 Sep 2009 05:41:49 PDT

We describe a novel, fundamental property of nucleobase structure, namely, pyramidilization at the N1/9 sites of purine and pyrimidine bases. Through a combined analyses of ultra-high-resolution X-ray structures of both oligonucleotides extracted from the Nucleic Acid Database and isolated nucleotides and nucleosides from the Cambridge Structural Database, together with a series of quantum chemical calculations, molecular dynamics (MD) simulations, and published solution nuclear magnetic resonance (NMR) data, we show that pyramidilization at the glycosidic nitrogen is an intrinsic property. This property is common to isolated nucleosides and nucleotides as well as oligonucleotides—it is also common to both RNA and DNA. Our analysis suggests that pyramidilization at N1/9 sites depends in a systematic way on the local structure of the nucleoside. Of note, the pyramidilization undergoes stereo-inversion upon reorientation of the glycosidic bond. The extent of the pyramidilization is further modulated by the conformation of the sugar ring. The observed pyramidilization is more pronounced for purine bases, while for pyrimidines it is negligible. We discuss how the assumption of nucleic acid base planarity can lead to systematic errors in determining the conformation of nucleotides from experimental data and from unconstrained MD simulations.

Nuclear export factor RBM15 facilitates the access of DBP5 to mRNA

Mon, 28 Sep 2009 05:41:44 PDT

The conserved mRNA export receptor NXF1 (Mex67 in yeast) assembles with messenger ribonucleoproteins (mRNP) in the nucleus and guides them through the nuclear pore complex into the cytoplasm. The DEAD family RNA helicase Dbp5 is essential for nuclear export of mRNA and is thought to dissociate Mex67 from mRNP upon translocation, thereby generating directional passage. However, the molecular mechanism by which Dbp5 recognizes Mex67-containing mRNP is not clear. Here we report that the human NXF1-binding protein RBM15 binds specifically to human DBP5 and facilitates its direct contact with mRNA in vivo. We found that RBM15 is targeted to the nuclear envelope, where it colocalizes extensively with DBP5 and NXF1. Gene silencing of RBM15 leads to cytoplasmic depletion and nuclear accumulation of general mRNA as well as individual endogenous transcripts, indicating that RBM15 is required for efficient mRNA export. We propose a model in which RBM15 acts locally at the nuclear pore complex, by facilitating the recognition of NXF1–mRNP complexes by DBP5 during translocation, thereby contributing to efficient mRNA export.

Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria

Mon, 28 Sep 2009 05:41:39 PDT

The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m¹A) and 3-methylcytosine (m³C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N⁶-ethenoadenine (A). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.