Nucleic Acids Research - Advance Access

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 topoisomerase I inhibition by camptothecin induces escape of RNA polymerase II from promoter-proximal pause site, antisense transcription and histone acetylation at the 5 human HIF-1{alpha} gene locus

Baranello, L., Bertozzi, D., Fogli, M. V., Pommier, Y., Capranico, G. — Fri, 23 Oct 2009 08:47:52 PDT

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.

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.

miRGen 2.0: a database of microRNA genomic information and regulation

Thu, 22 Oct 2009 08:45:21 PDT

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://diana.cslab.ece.ntua.gr/miRGen/.

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.

Fine-grained annotation and classification of de novo predicted LTR retrotransposons

Steinbiss, S., Willhoeft, U., Gremme, G., Kurtz, S. — Mon, 28 Sep 2009 05:41:28 PDT

Long terminal repeat (LTR) retrotransposons and endogenous retroviruses (ERVs) are transposable elements in eukaryotic genomes well suited for computational identification. De novo identification tools determine the position of potential LTR retrotransposon or ERV insertions in genomic sequences. For further analysis, it is desirable to obtain an annotation of the internal structure of such candidates. This article presents LTRdigest, a novel software tool for automated annotation of internal features of putative LTR retrotransposons. It uses local alignment and hidden Markov model-based algorithms to detect retrotransposon-associated protein domains as well as primer binding sites and polypurine tracts. As an example, we used LTRdigest results to identify 88 (near) full-length ERVs in the chromosome 4 sequence of Mus musculus, separating them from truncated insertions and other repeats. Furthermore, we propose a work flow for the use of LTRdigest in de novo LTR retrotransposon classification and perform an exemplary de novo analysis on the Drosophila melanogaster genome as a proof of concept. Using a new method solely based on the annotations generated by LTRdigest, 518 potential LTR retrotransposons were automatically assigned to 62 candidate groups. Representative sequences from 41 of these 62 groups were matched to reference sequences with >80% global sequence similarity.

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Mon, 28 Sep 2009 05:41:17 PDT

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA-K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE-mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C–rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism.

The 3'-5' proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases

Russell, H. J., Richardson, T. T., Emptage, K., Connolly, B. A. — Sat, 26 Sep 2009 02:07:03 PDT

Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3'–5' proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3' base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3'–5' proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases.

EMMA--mouse mutant resources for the international scientific community

Sat, 26 Sep 2009 02:07:01 PDT

The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org.

FSscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots

Liao, P.-Y., Choi, Y. S., Lee, K. H. — Sat, 26 Sep 2009 02:06:57 PDT

In +1 programmed ribosomal frameshifting (PRF), ribosomes skip one nucleotide toward the 3'-end during translation. Most of the genes known to demonstrate +1 PRF have been discovered by chance or by searching homologous genes. Here, a bioinformatic framework called FSscan is developed to perform a systematic search for potential +1 frameshift sites in the Escherichia coli genome. Based on a current state of the art understanding of the mechanism of +1 PRF, FSscan calculates scores for a 16-nt window along a gene sequence according to different effects of the stimulatory signals, and ribosome E-, P- and A-site interactions. FSscan successfully identified the +1 PRF site in prfB and predicted yehP, pepP, nuoE and cheA as +1 frameshift candidates in the E. coli genome. Empirical results demonstrated that potential +1 frameshift sequences identified promoted significant levels of +1 frameshifting in vivo. Mass spectrometry analysis confirmed the presence of the frameshifted proteins expressed from a yehP-egfp fusion construct. FSscan allows a genome-wide and systematic search for +1 frameshift sites in E. coli. The results have implications for bioinformatic identification of novel frameshift proteins, ribosomal frameshifting, coding sequence detection and the application of mass spectrometry on studying frameshift proteins.

The single polypeptide restriction-modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops

Smith, R. M., Josephsen, J., Szczelkun, M. D. — Sat, 26 Sep 2009 02:06:55 PDT

To cleave DNA, the single polypeptide restriction–modification enzyme LlaGI must communicate between a pair of indirectly repeated recognition sites. We demonstrate that this communication occurs by a 1-dimensional route, namely unidirectional dsDNA loop translocation rightward of the specific recognition sequence 5'-CTnGAyG-3' as written (where n is either A, G, C or T and y is either C or T). Motion across thousands of base pairs is catalysed by the helicase domain and requires the hydrolysis of 1.5-2 ATP per base pair. DNA loop extrusion is accompanied by changes in DNA twist consistent with the motor following the helical pitch of the polynucleotide track. LlaGI is therefore an example of a polypeptide that is a completely self-contained, multi-functional molecular machine.

The HSV-1 ICP27 RGG box specifically binds flexible, GC-rich sequences but not G-quartet structures

Corbin-Lickfett, K. A., Chen, I-H. B., Cocco, M. J., Sandri-Goldin, R. M. — Sat, 26 Sep 2009 02:06:53 PDT

Herpes simplex virus 1 (HSV-1) protein ICP27, an important regulator for viral gene expression, directly recognizes and exports viral RNA through an N-terminal RGG box RNA binding motif, which is necessary and sufficient for RNA binding. An ICP27 N-terminal peptide, including the RGG box RNA binding motif, was expressed and its binding specificity was analyzed using EMSA and SELEX. DNA oligonucleotides corresponding to HSV-1 glycoprotein C (gC) mRNA, identified in a yeast three-hybrid analysis, were screened for binding to the ICP27 N-terminal peptide in EMSA experiments. The ICP27 N-terminus was able to bind most gC substrates. Notably, the ICP27 RGG box was unable to bind G-quartet structures recognized by the RGG domains of other proteins. SELEX analysis identified GC-rich RNA sequences as a common feature of recognition. NMR analysis of SELEX and gC sequences revealed that sequences able to bind to ICP27 did not form secondary structures and conversely, sequences that were not able to bind to ICP27 gave spectra consistent with base-pairing. Therefore, the ICP27 RGG box is unique in its recognition of nucleic acid sequences compared to other RGG box proteins; it prefers flexible, GC-rich substrates that do not form stable secondary structures.

Ion-induced folding of a kink turn that departs from the conventional sequence

Schroeder, K. T., Lilley, D. M. J. — Sat, 26 Sep 2009 02:06:50 PDT

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G•A and A•G base pairs, and when these G•A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A•G pair is replaced by a non-Watson–Crick A•U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg²⁺ or Na⁺ ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A•G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson–Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A•U pair is more important than the other.

Recode-2: new design, new search tools, and many more genes

Fri, 25 Sep 2009 22:39:27 PDT

‘Recoding’ is a term used to describe non-standard read-out of the genetic code, and encompasses such phenomena as programmed ribosomal frameshifting, stop codon readthrough, selenocysteine insertion and translational bypassing. Although only a small proportion of genes utilize recoding in protein synthesis, accurate annotation of ‘recoded’ genes lags far behind annotation of ‘standard’ genes. In order to address this issue, provide a service to researchers in the field, and offer training data for developers of gene-annotation software, we have gathered together known cases of recoding within the Recode database. Recode-2 is an improved and updated version of the database. It provides access to detailed information on genes known to utilize translational recoding and allows complex search queries, browsing of recoding data and enhanced visualization of annotated sequence elements. At present, the Recode-2 database stores information on approximately 1500 genes that are known to utilize recoding in their expression—a factor of approximately three increase over the previous version of the database. Recode-2 is available at http://recode.ucc.ie

tRNA over-expression in breast cancer and functional consequences

Pavon-Eternod, M., Gomes, S., Geslain, R., Dai, Q., Rosner, M. R., Pan, T. — Fri, 25 Sep 2009 22:39:25 PDT

Increased proliferation and elevated levels of protein synthesis are characteristics of transformed and tumor cells. Though components of the translation machinery are often misregulated in cancers, what role tRNA plays in cancer cells has not been explored. We compare genome-wide tRNA expression in cancer-derived versus non-cancer-derived breast cell lines, as well as tRNA expression in breast tumors versus normal breast tissues. In cancer-derived versus non-cancer-derived cell lines, nuclear-encoded tRNAs increase by up to 3-fold and mitochondrial-encoded tRNAs increase by up to 5-fold. In tumors versus normal breast tissues, both nuclear- and mitochondrial-encoded tRNAs increase up to 10-fold. This tRNA over-expression is selective and coordinates with the properties of cognate amino acids. Nuclear- and mitochondrial-encoded tRNAs exhibit distinct expression patterns, indicating that tRNAs can be used as biomarkers for breast cancer. We also performed association analysis for codon usage-tRNA expression for the cell lines. tRNA isoacceptor expression levels are not geared towards optimal translation of house-keeping or cell line specific genes. Instead, tRNA isoacceptor expression levels may favor the translation of cancer-related genes having regulatory roles. Our results suggest a functional consequence of tRNA over-expression in tumor cells. tRNA isoacceptor over-expression may increase the translational efficiency of genes relevant to cancer development and progression.

PDBselect 1992-2009 and PDBfilter-select

Griep, S., Hobohm, U. — Fri, 25 Sep 2009 22:39:22 PDT

PDBselect (http://bioinfo.tg.fh-giessen.de/pdbselect/) is a list of representative protein chains with low mutual sequence identity selected from the protein data bank (PDB) to enable unbiased statistics. The list increased from 155 chains in 1992 to more than 4500 chains in 2009. PDBfilter-select is an online service to generate user-defined selections.

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Shakirov, E. V., Song, X., Joseph, J. A., Shippen, D. E. — Fri, 25 Sep 2009 22:39:20 PDT

Telomeric DNA terminates with a single-stranded 3' G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.

MiCroKit 3.0: an integrated database of midbody, centrosome and kinetochore

Fri, 25 Sep 2009 22:39:17 PDT

During cell division/mitosis, a specific subset of proteins is spatially and temporally assembled into protein super complexes in three distinct regions, i.e. centrosome/spindle pole, kinetochore/centromere and midbody/cleavage furrow/phragmoplast/bud neck, and modulates cell division process faithfully. Although many experimental efforts have been carried out to investigate the characteristics of these proteins, no integrated database was available. Here, we present the MiCroKit database (http://microkit.biocuckoo.org) of proteins that localize in midbody, centrosome and/or kinetochore. We collected into the MiCroKit database experimentally verified microkit proteins from the scientific literature that have unambiguous supportive evidence for subcellular localization under fluorescent microscope. The current version of MiCroKit 3.0 provides detailed information for 1489 microkit proteins from seven model organisms, including Saccharomyces cerevisiae, Schizasaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, Mus musculus and Homo sapiens. Moreover, the orthologous information was provided for these microkit proteins, and could be a useful resource for further experimental identification. The online service of MiCroKit database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0).

SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin

Fri, 25 Sep 2009 22:39:15 PDT

SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.

Crystal structure of the EndoG/EndoGI complex: mechanism of EndoG inhibition

Loll, B., Gebhardt, M., Wahle, E., Meinhart, A. — Fri, 25 Sep 2009 22:39:07 PDT

EndoG is a ubiquitous nuclease that is translocated into the nucleus during apoptosis to participate in DNA degradation. The enzyme cleaves double- and single-stranded DNA and RNA. Related nucleases are found in eukaryotes and prokaryotes, which have evolved sophisticated mechanisms for genome protection against self-antagonizing nuclease activity. Common mechanisms of inhibition are secretion, sequestration into a separate cellular compartment or by binding to protein inhibitors. Although EndoG is silenced by compartmentalization into the mitochondrial intermembrane space, a nucleus-localized protein inhibitor protects cellular polynucleotides from degradation by stray EndoG under non-apoptotic conditions in Drosophila. Here, we report the first three-dimensional structure of EndoG in complex with its inhibitor EndoGI. Although the mechanism of inhibition is reminiscent of bacterial protein inhibitors, EndoGI has evolved independently from a generic protein-protein interaction module. EndoGI is a two-domain protein that binds the active sites of two monomers of EndoG, with EndoG being sandwiched between EndoGI. Since the amino acid sequences of eukaryotic EndoG homologues are highly conserved, this model is valid for eukaryotic dimeric EndoG in general. The structure indicates that the two active sites of EndoG occupy the most remote spatial position possible at the molecular surface and a concerted substrate processing is unlikely.

Solid polymeric microparticles enhance the delivery of siRNA to macrophages in vivo

Lee, S., Yang, S. C., Kao, C.-Y., Pierce, R. H., Murthy, N. — Fri, 25 Sep 2009 22:39:04 PDT

Therapeutics based on small interfering RNA (siRNA) have a great clinical potential; however, delivery problems have limited their clinical efficacy, and new siRNA delivery vehicles are greatly needed. In this report, we demonstrate that submicron particles (800–900 nm) composed of the polyketal PK3 and chloroquine, termed as the PKCNs, can deliver tumor necrosis factor- (TNF-) siRNA in vivo to Kupffer cells efficiently and inhibit gene expression in the liver at concentrations as low as 3.5 µg/kg. The high delivery efficiency of the PKCNs arises from the unique properties of PK3, which can protect siRNA from serum nucleases, stimulate cell uptake and trigger a colloid osmotic disruption of the phagosome and release encapsulated siRNA into the cell cytoplasm. We anticipate numerous applications of the PKCNs for siRNA delivery to macrophages, given their high delivery efficiency, and the central role of macrophages in causing diseases such as hepatitis, liver cirrhosis and chronic renal disease.

RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

Park, S. W., Huang, W.-H., Persaud, S. D., Wei, L.-N. — Wed, 23 Sep 2009 22:18:01 PDT

Cellular retinoic acid binding protein 1 (Crabp1) gene is biphasically (proliferation versus differentiation) regulated by thyroid hormone (T3) in 3T3-L1 cells. This study examines T3-repression of Crabp1 gene during adipocyte differentiation. T3 repression of Crabp1 requires receptor interacting protein 140 (RIP140). During differentiation, the juxtaposed chromatin configuration of Crabp1 promoter with its upstream region is maintained, but the 6-nucleosomes spanning thyroid hormone response element to transcription initiation site slide bi-directionally, with the third nucleosome remaining at the same position throughout differentiation. On the basal promoter, RIP140 replaces coactivators GRIP1 and PCAF and forms a repressive complex with CtBP1, HDAC3 and G9a. Initially active chromatin marks on this promoter, histone modifications H3-Ac and H3K4-me3, are weakened whereas repressive chromatin marks, H3K9-me3 and H3K27-me3 modification and recruitment of G9a, HP1, HP1 and H1, are intensified. This is the first study to examine chromatin remodeling, during the phase of hormone repression, of a bi-directionally regulated hormone target gene, and provides evidence for a functional role of RIP140 in chromatin remodeling to repress hormone target gene expression.

Two Dot1 isoforms in Saccharomyces cerevisiae as a result of leaky scanning by the ribosome

Wed, 23 Sep 2009 22:17:58 PDT

Dot1 is a conserved histone methyltransferase that methylates histone H3 on lysine 79. We previously observed that in Saccharomyces cerevisiae, a single DOT1 gene encodes two Dot1 protein species. Here, we show that the relative abundance of the two isoforms changed under nutrient-limiting conditions. A mutagenesis approach showed that the two Dot1 isoforms are produced from two alternative translation start sites as a result of leaky scanning by the ribosome. The leaky scanning was not affected by the 5'- or 3'-untranslated regions of DOT1, indicating that translation initiation is determined by the DOT1 coding sequence. Construction of yeast strains expressing either one of the isoforms showed that both were sufficient for Dot1’s role in global H3K79 methylation and telomeric gene silencing. However, the absence of the long isoform of Dot1 altered the resistance of yeast cells to the chitin-binding drug Calcofluor White, suggesting that the two Dot1 isoforms have a differential function in cell wall biogenesis.

Universal function-specificity of codon usage

Najafabadi, H. S., Goodarzi, H., Salavati, R. — Tue, 22 Sep 2009 09:13:05 PDT

Synonymous codon usage has long been known as a factor that affects average expression level of proteins in fast-growing microorganisms, but neither its role in dynamic changes of expression in response to environmental changes nor selective factors shaping it in the genomes of higher eukaryotes have been fully understood. Here, we propose that codon usage is ubiquitously selected to synchronize the translation efficiency with the dynamic alteration of protein expression in response to environmental and physiological changes. Our analysis reveals that codon usage is universally correlated with gene function, suggesting its potential contribution to synchronized regulation of genes with similar functions. We directly show that coexpressed genes have similar synonymous codon usages within the genomes of human, yeast, Caenorhabditis elegans and Escherichia coli. We also demonstrate that perturbing the codon usage directly affects the level or even direction of changes in protein expression in response to environmental stimuli. Perturbing tRNA composition also has tangible phenotypic effects on the cell. By showing that codon usage is universally function-specific, our results expand, to almost all organisms, the notion that cells may need to dynamically alter their intracellular tRNA composition in order to adapt to their new environment or physiological role.

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

Pastor, T., Talotti, G., Lewandowska, M. A., Pagani, F. — Tue, 22 Sep 2009 09:13:01 PDT

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.

The transcriptional co-activator PCAF regulates cdk2 activity

Tue, 22 Sep 2009 09:12:57 PDT

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G₂/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G₂/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

A trimeric DNA polymerase complex increases the native replication processivity

Mikheikin, A. L., Lin, H.-K., Mehta, P., Jen-Jacobson, L., Trakselis, M. A. — Tue, 22 Sep 2009 09:12:49 PDT

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase–polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.

The Aspergillus Genome Database, a curated comparative genomics resource for gene, protein and sequence information for the Aspergillus research community

Tue, 22 Sep 2009 09:12:45 PDT

The Aspergillus Genome Database (AspGD) is an online genomics resource for researchers studying the genetics and molecular biology of the Aspergilli. AspGD combines high-quality manual curation of the experimental scientific literature examining the genetics and molecular biology of Aspergilli, cutting-edge comparative genomics approaches to iteratively refine and improve structural gene annotations across multiple Aspergillus species, and web-based research tools for accessing and exploring the data. All of these data are freely available at http://www.aspgd.org. We welcome feedback from users and the research community at aspergillus-curator@genome.stanford.edu.

HRTBLDb: an informative data resource for hormone receptors target binding loci

Kennedy, B. A., Gao, W., Huang, T. H.-M., Jin, V. X. — Tue, 22 Sep 2009 09:12:41 PDT

Three hormone receptors, the estrogen receptor (ER), the androgen receptor (AR) and glucocorticoid receptor (GR) play an important role in regulating the cellular differentiation tissue development of skin, bone, the brain and the endocrine system; therefore, there is a strong scientific need to identify and characterize hormone receptor transcriptional regulation. Given that the vast amount of regulatory data for hormone being produced by ChIP-based high-throughput experiments is widely scattered in disparate, poorly cross-indexed data stores, a flexible platform for organizing and relating these data would provide significant value. We created a data management system called the Hormone Receptor Target Binding Loci, HRTBLDb (http://motif.bmi.ohio-state.edu/hrtbldb), to address this problem. This database contains hormone receptor binding regions (binding loci) from in vivo ChIP-based high-throughput experiments as well as in silico, computationally predicted, binding motifs and cis-regulatory modules for the co-occurring transcription factor binding motifs, which are within a binding locus. It also contains individual binding sites whose regulatory action has been verified by in vitro experiments. The current version contains 44 673 binding elements with 114 hormone response elements which are verified by in vitro experiments; 75 binding motifs which occur with a hormone response element and whose co-regulatory action is verified by in vitro experiments; 18 472 binding loci from in vivo experiments; and 26 012 computationally predicted binding motifs.

A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation

Korolev, N., Berezhnoy, N. V., Eom, K. D., Tam, J. P., Nordenskiold, L. — Tue, 22 Sep 2009 09:12:37 PDT

We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear -oligo(l-lysines), (denoted Kn, n = 3–10, 31; n is the number of lysines equal to the ligand charge) and branched -substituted homologues of K10: YK10, LK10 (Z = +10); RK10, YRK10 and LYRK10 (Z = +20). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, C_KCl). The dependence of EC₅₀ (ligand concentration at the midpoint of DNA condensation) on C_KCl shows the existence of a salt-independent regime at low C_KCl and a salt-dependent regime with a steep rise of EC₅₀ with increase of C_KCl. Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher C_KCl. A novel and simple relationship describing the EC₅₀ dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand–DNA complex is derived. For the -oligolysines K3–K10, the experimental dependencies of EC₅₀ on C_KCl and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed.

3D-footprint: a database for the structural analysis of protein-DNA complexes

Contreras-Moreira, B. — Fri, 18 Sep 2009 22:40:04 PDT

3D-footprint is a living database, updated and curated on a weekly basis, which provides estimates of binding specificity for all protein–DNA complexes available at the Protein Data Bank. The web interface allows the user to: (i) browse DNA-binding proteins by keyword; (ii) find proteins that recognize a similar DNA motif and (iii) BLAST similar DNA-binding proteins, highlighting interface residues in the resulting alignments. Each complex in the database is dissected to draw interface graphs and footprint logos, and two complementary algorithms are employed to characterize binding specificity. Moreover, oligonucleotide sequences extracted from literature abstracts are reported in order to show the range of variant sites bound by each protein and other related proteins. Benchmark experiments, including comparisons with expert-curated databases RegulonDB and TRANSFAC, support the quality of structure-based estimates of specificity. The relevant content of the database is available for download as flat files and it is also possible to use the 3D-footprint pipeline to analyze protein coordinates input by the user. 3D-footprint is available at http://floresta.eead.csic.es/3dfootprint with demo buttons and a comprehensive tutorial that illustrates the main uses of this resource.

The University of Minnesota Biocatalysis/Biodegradation Database: improving public access

Gao, J., Ellis, L. B. M., Wackett, L. P. — Fri, 18 Sep 2009 22:40:02 PDT

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.msi.umn.edu/) began in 1995 and now contains information on almost 1200 compounds, over 800 enzymes, almost 1300 reactions and almost 500 microorganism entries. Besides these data, it includes a Biochemical Periodic Table (UM-BPT) and a rule-based Pathway Prediction System (UM-PPS) (http://umbbd.msi.umn.edu/predict/) that predicts plausible pathways for microbial degradation of organic compounds. Currently, the UM-PPS contains 260 biotransformation rules derived from reactions found in the UM-BBD and scientific literature. Public access to UM-BBD data is increasing. UM-BBD compound data are now contributed to PubChem and ChemSpider, the public chemical databases. A new mirror website of the UM-BBD, UM-BPT and UM-PPS is being developed at ETH Zürich to improve speed and reliability of online access from anywhere in the world.

EMAGE mouse embryo spatial gene expression database: 2010 update

Fri, 18 Sep 2009 22:40:00 PDT

EMAGE (http://www.emouseatlas.org/emage) is a freely available online database of in situ gene expression patterns in the developing mouse embryo. Gene expression domains from raw images are extracted and integrated spatially into a set of standard 3D virtual mouse embryos at different stages of development, which allows data interrogation by spatial methods. An anatomy ontology is also used to describe sites of expression, which allows data to be queried using text-based methods. Here, we describe recent enhancements to EMAGE including: the release of a completely re-designed website, which offers integration of many different search functions in HTML web pages, improved user feedback and the ability to find similar expression patterns at the click of a button; back-end refactoring from an object oriented to relational architecture, allowing associated SQL access; and the provision of further access by standard formatted URLs and a Java API. We have also increased data coverage by sourcing from a greater selection of journals and developed automated methods for spatial data annotation that are being applied to spatially incorporate the genome-wide (~19 000 gene) ‘EURExpress’ dataset into EMAGE.

Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions

Fri, 18 Sep 2009 22:39:57 PDT

The wobble uridine of certain bacterial and mitochondrial tRNAs is modified, at position 5, through an unknown reaction pathway that utilizes the evolutionarily conserved MnmE and GidA proteins. The resulting modification (a methyluridine derivative) plays a critical role in decoding NNG/A codons and reading frame maintenance during mRNA translation. The lack of this tRNA modification produces a pleiotropic phenotype in bacteria and has been associated with mitochondrial encephalomyopathies in humans. In this work, we use in vitro and in vivo approaches to characterize the enzymatic pathway controlled by the Escherichia coli MnmE•GidA complex. Surprisingly, this complex catalyzes two different GTP- and FAD-dependent reactions, which produce 5-aminomethyluridine and 5-carboxymethylamino-methyluridine using ammonium and glycine, respectively, as substrates. In both reactions, methylene-tetrahydrofolate is the most probable source to form the C5-methylene moiety, whereas NADH is dispensable in vitro unless FAD levels are limiting. Our results allow us to reformulate the bacterial MnmE•GidA dependent pathway and propose a novel mechanism for the modification reactions performed by the MnmE and GidA family proteins.

A SRS2 homolog from Arabidopsis thaliana disrupts recombinogenic DNA intermediates and facilitates single strand annealing

Blanck, S., Kobbe, D., Hartung, F., Fengler, K., Focke, M., Puchta, H. — Fri, 18 Sep 2009 22:39:54 PDT

Genetic and biochemical analyses of SRS2 homologs in fungi indicate a function in the processing of homologous recombination (HR) intermediates. To date, no SRS2 homologs have been described and analyzed in higher eukaryotes. Here, we report the first biochemical characterization of an SRS2 homolog from a multicellular eukaryote, the plant Arabidopsis thaliana. We studied the basic properties of AtSRS2 and were able to show that it is a functional 3'- to 5'-helicase. Furthermore, we characterized its biochemical function on recombinogenic intermediates and were able to show the unwinding of nicked Holliday junctions (HJs) and partial HJs (PX junctions). For the first time, we demonstrated strand annealing activity for an SRS2 homolog and characterized its strand pairing activity in detail. Our results indicate that AtSRS2 has properties that enable it to be involved in different steps during the processing of recombination intermediates. On the one hand, it could be involved in the unwinding of an elongating invading strand from a donor strand, while on the other hand, it could be involved in the annealing of the elongated strand at a later step.

Demonstration of all-or-none loss of imprinting in mRNA expression in single cells

Fri, 18 Sep 2009 22:39:52 PDT

Loss of imprinting (LOI) is the reactivation of the silenced allele of an imprinted gene, leading to perturbation of monoallelic expression. We tested the hypothesis that LOI of PLAGL1, a representative maternally imprinted gene, occurs through an all-or-none process leading to a mixture of fully imprinted and nonimprinted cells. Herein using a quantitative RT-PCR-based experimental approach, we measured LOI at the single cell level in human trophoblasts and demonstrated a broad distribution of LOI among cells exhibiting LOI, with the mean centered at ~100% LOI. There was a significant (P < 0.01) increase in expression after 2 days of 5-aza-2'-deoxycytidine (AZA) treatment and a significant (P < 0.01) increase in LOI after both 1 and 2 days of AZA treatment, while the distribution remained broad and centered at ~100% LOI. We propose a transcriptional pulsing model to show that the broadness of the distribution reflects the stochastic nature of expression between the two alleles in each cell. The mean of the distribution of LOI in the cells is consistent with our hypothesis that LOI occurs by an all-or-none process. All-or-none LOI could lead to a second distinct cell population that may have a selective advantage, leading to variation of LOI in normal tissues, such as the placenta, or in neoplastic cells.

Primary sequence and epigenetic determinants of in vivo occupancy of genomic DNA by GATA1

Fri, 18 Sep 2009 22:39:49 PDT

DNA sequence motifs and epigenetic modifications contribute to specific binding by a transcription factor, but the extent to which each feature determines occupancy in vivo is poorly understood. We addressed this question in erythroid cells by identifying DNA segments occupied by GATA1 and measuring the level of trimethylation of histone H3 lysine 27 (H3K27me3) and monomethylation of H3 lysine 4 (H3K4me1) along a 66 Mb region of mouse chromosome 7. While 91% of the GATA1-occupied segments contain the consensus binding-site motif WGATAR, only ~0.7% of DNA segments with such a motif are occupied. Using a discriminative motif enumeration method, we identified additional motifs predictive of occupancy given the presence of WGATAR. The specific motif variant AGATAA and occurrence of multiple WGATAR motifs are both strong discriminators. Combining motifs to pair a WGATAR motif with a binding site motif for GATA1, EKLF or SP1 improves discriminative power. Epigenetic modifications are also strong determinants, with the factor-bound segments highly enriched for H3K4me1 and depleted of H3K27me3. Combining primary sequence and epigenetic determinants captures 52% of the GATA1-occupied DNA segments and substantially increases the specificity, to one out of seven segments with the required motif combination and epigenetic signals being bound.

Phylogenetic screening of a bacterial, metagenomic library using homing endonuclease restriction and marker insertion

Yung, P. Y., Burke, C., Lewis, M., Egan, S., Kjelleberg, S., Thomas, T. — Fri, 18 Sep 2009 22:40:07 PDT

Metagenomics provides access to the uncultured majority of the microbial world. The approaches employed in this field have, however, had limited success in linking functional genes to the taxonomic or phylogenetic origin of the organism they belong to. Here we present an efficient strategy to recover environmental DNA fragments that contain phylogenetic marker genes from metagenomic libraries. Our method involves the cleavage of 23S ribsosmal RNA (rRNA) genes within pooled library clones by the homing endonuclease I-CeuI followed by the insertion and selection of an antibiotic resistance cassette. This approach was applied to screen a library of 6500 fosmid clones derived from the microbial community associated with the sponge Cymbastela concentrica. Several fosmid clones were recovered after the screen and detailed phylogenetic and taxonomic assignment based on the rRNA gene showed that they belong to previously unknown organisms. In addition, compositional features of these fosmid clones were used to classify and taxonomically assign a dataset of environmental shotgun sequences. Our approach represents a valuable tool for the analysis of rapidly increasing, environmental DNA sequencing information.

Influence of local sequence context on damaged base conformation in human DNA polymerase {iota}: molecular dynamics studies of nucleotide incorporation opposite a benzo[a]pyrene-derived adenine lesion

Donny-Clark, K., Broyde, S. — Fri, 18 Sep 2009 22:39:47 PDT

Human DNA polymerase is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions in both near error-free and error-prone bypass. With undamaged templating purines polymerase normally favors Hoogsteen base pairing. Polymerase can incorporate nucleotides opposite a benzo[a]pyrene-derived adenine lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. We performed molecular modeling and molecular dynamics simulations to elucidate the structural basis for lesion bypass. Our results suggest that hydrogen bonds between the benzo[a]pyrenyl moiety and nearby bases limit the movement of the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5'-CAGA*TT-3' sequence. This facilitates correct incorporation of dT via a Watson–Crick pair. In a 5'-TTTA*GA-3' sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA*, there is also an opportunity for increased misincorporation of dGTP. These results expand our understanding of the versatility and flexibility of polymerase and its lesion bypass functions in humans.

Impact of DNA-binding position variants on yeast gene expression

Swamy, K. B. S., Cho, C.-Y., Chiang, S., Tsai, Z. T.-Y., Tsai, H.-K. — Fri, 18 Sep 2009 22:39:44 PDT

Transcription factors (TFs) regulate gene expression by binding to specific binding sites (TFBSs) in gene promoters. TFBS motifs may contain one or more variable positions. Although the prevailing assumption is that nucleotide variants at such positions are functionally equivalent, there is increasing evidence that such variants play a role in regulation of gene expression. In this article, we propose a method for studying the relationship between the expression of target genes and nucleotide variants in TFBS motifs at a genome-wide scale in Saccharomyces cerevisiae, especially the combinatorial effects of variants at two positions. Our analysis shows that nucleotide variations in more than one-third of variable positions and in 20% of dependent position pairs are highly correlated to gene expression. We define such positions as ‘functional’. However, some positions are only functional as dependent pairs, but not individually. In addition, a significant proportion of the functional positions have been well conserved across all yeast-related species studied. We also find that some positions require the presence of co-occurring TFs, while others maintain their functionality in the absence of a co-occurring TF. Our analysis supports the importance of nucleotide variants at variable positions of TFBSs in gene regulation.

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 'Slicer' independent and can be mediated by Ago1

Fri, 18 Sep 2009 22:39:41 PDT

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the ‘off-target’ mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated.

Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny

Fri, 18 Sep 2009 22:39:39 PDT

Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNA^Asp in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNA^Asp including predominantly the cloverleaf. On the contrary, the native tRNA^Asp folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis–Westhof interactions, the tertiary network core building rules apply to all tRNA^Asp from mammalian mitochondria.

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant

Fri, 18 Sep 2009 22:39:36 PDT

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA. Inspection of the inter atomic distances in proximity of the active site shows that in the mutant the distance between the amino group of Lys532 side chain and the 5' OH of the scissile phosphate is longer than the wild type enzyme, providing an atomic explanation for the reduced religation rate of the mutant. Taken together these results indicate the existence of a long range communication between the linker domain and the active site region and points out the crucial role of the linker in the modulation of the catalytic activity.

MGEScan-non-LTR: computational identification and classification of autonomous non-LTR retrotransposons in eukaryotic genomes

Rho, M., Tang, H. — Thu, 17 Sep 2009 08:19:34 PDT

Computational methods for genome-wide identification of mobile genetic elements (MGEs) have become increasingly necessary for both genome annotation and evolutionary studies. Non-long terminal repeat (non-LTR) retrotransposons are a class of MGEs that have been found in most eukaryotic genomes, sometimes in extremely high numbers. In this article, we present a computational tool, MGEScan-non-LTR, for the identification of non-LTR retrotransposons in genomic sequences, following a computational approach inspired by a generalized hidden Markov model (GHMM). Three different states represent two different protein domains and inter-domain linker regions encoded in the non-LTR retrotransposons, and their scores are evaluated by using profile hidden Markov models (for protein domains) and Gaussian Bayes classifiers (for linker regions), respectively. In order to classify the non-LTR retrotransposons into one of the 12 previously characterized clades using the same model, we defined separate states for different clades. MGEScan-non-LTR was tested on the genome sequences of four eukaryotic organisms, Drosophila melanogaster, Daphnia pulex, Ciona intestinalis and Strongylocentrotus purpuratus. For the D. melanogaster genome, MGEScan-non-LTR found all known ‘full-length’ elements and simultaneously classified them into the clades CR1, I, Jockey, LOA and R1. Notably, for the D. pulex genome, in which no non-LTR retrotransposon has been annotated, MGEScan-non-LTR found a significantly larger number of elements than did RepeatMasker, using the current version of the RepBase Update library. We also identified novel elements in the other two genomes, which have only been partially studied for non-LTR retrotransposons.

DNA translocation activity of the multifunctional replication protein ORF904 from the archaeal plasmid pRN1

Sanchez, M., Drechsler, M., Stark, H., Lipps, G. — Thu, 17 Sep 2009 08:19:30 PDT

The replication protein ORF904 from the plasmid pRN1 is a multifunctional enzyme with ATPase-, primase- and DNA polymerase activity. Sequence analysis suggests the presence of at least two conserved domains: an N-terminal prim/pol domain with primase and DNA polymerase activities and a C-terminal superfamily 3 helicase domain with a strong double-stranded DNA dependant ATPase activity. The exact molecular function of the helicase domain in the process of plasmid replication remains unclear. Potentially this motor protein is involved in duplex remodelling and/or origin opening at the plasmid replication origin. In support of this we found that the monomeric replication protein ORF904 forms a hexameric ring in the presence of DNA. It is able to translocate along single-stranded DNA in 3'–5' direction as well as on double-stranded DNA. Critical residues important for ATPase activity and DNA translocation activity were identified and are in agreement with a homology model of the helicase domain. In addition we propose that a winged helix DNA-binding domain at the C-terminus of the helicase domain could assist the binding of the replication protein specifically to the replication origin.

Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells

Covo, S., de Villartay, J.-P., Jeggo, P. A., Livneh, Z. — Thu, 17 Sep 2009 08:19:27 PDT

Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5' overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ.

FIGfams: yet another set of protein families

Meyer, F., Overbeek, R., Rodriguez, A. — Thu, 17 Sep 2009 08:19:24 PDT

We present FIGfams, a new collection of over 100 000 protein families that are the product of manual curation and close strain comparison. Using the Subsystem approach the manual curation is carried out, ensuring a previously unattained degree of throughput and consistency. FIGfams are based on over 950 000 manually annotated proteins and across many hundred Bacteria and Archaea. Associated with each FIGfam is a two-tiered, rapid, accurate decision procedure to determine family membership for new proteins. FIGfams are freely available under an open source license. These can be downloaded at ftp://ftp.theseed.org/FIGfams/. The web site for FIGfams is http://www.theseed.org/wiki/FIGfams/

The Staphylococcus aureus pSK41 plasmid-encoded ArtA protein is a master regulator of plasmid transmission genes and contains a RHH motif used in alternate DNA-binding modes

Wed, 16 Sep 2009 08:45:52 PDT

Plasmids harbored by Staphylococcus aureus are a major contributor to the spread of bacterial multi-drug resistance. Plasmid conjugation and partition are critical to the dissemination and inheritance of such plasmids. Here, we demonstrate that the ArtA protein encoded by the S. aureus multi-resistance plasmid pSK41 is a global transcriptional regulator of pSK41 genes, including those involved in conjugation and segregation. ArtA shows no sequence homology to any structurally characterized DNA-binding protein. To elucidate the mechanism by which it specifically recognizes its DNA site, we obtained the structure of ArtA bound to its cognate operator, ACATGACATG. The structure reveals that ArtA is representative of a new family of ribbon–helix–helix (RHH) DNA-binding proteins that contain extended, N-terminal basic motifs. Strikingly, unlike most well-studied RHH proteins ArtA binds its cognate operators as a dimer. However, we demonstrate that it is also able to recognize an atypical operator site by binding as a dimer-of-dimers and the extended N-terminal regions of ArtA were shown to be essential for this dimer-of-dimer binding mode. Thus, these data indicate that ArtA is a master regulator of genes critical for both horizontal and vertical transmission of pSK41 and that it can recognize DNA utilizing alternate binding modes.

Involvement of p54(nrb), a PSF partner protein, in DNA double-strand break repair and radioresistance

Li, S., Kuhne, W. W., Kulharya, A., Hudson, F. Z., Ha, K., Cao, Z., Dynan, W. S. — Wed, 16 Sep 2009 08:45:47 PDT

Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a -H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.

RNA-protein binding kinetics in an automated microfluidic reactor

Ridgeway, W. K., Seitaridou, E., Phillips, R., Williamson, J. R. — Wed, 16 Sep 2009 08:45:44 PDT

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

Du, Z., Zhao, Y., Li, N. — Wed, 16 Sep 2009 08:45:41 PDT

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure.

Transcription from bacteriophage {lambda} pR promoter is regulated independently and antagonistically by DksA and ppGpp

Lyzen, R., Kochanowska, M., Wegrzyn, G., Szalewska-Palasz, A. — Wed, 16 Sep 2009 08:45:38 PDT

The stringent response effector, guanosine tetraphosphate (ppGpp), adjust gene expression and physiology in bacteria, by affecting the activity of various promoters. RNA polymerase-interacting protein, DksA, was proposed to be the co-factor of ppGpp effects; however, there are reports suggesting independent roles of these regulators. Bacteriophage major lytic promoter, pR, is down-regulated by the stringent response and ppGpp. Here, we present evidence that DksA significantly stimulates pR-initiated transcription in vitro in the reconstituted system. DksA is also indispensable for pR activity in vivo. DksA-mediated activation of pR-initiated transcription is predominant over ppGpp effects in the presence of both regulators in vitro. The possible role of the opposite regulation by ppGpp and DksA in phage development is discussed. The major mechanism of DksA-mediated activation of transcription from pR involves facilitating of RNA polymerase binding to the promoter region, which results in more productive transcription initiation. Thus, our results provide evidence for the first promoter inhibited by ppGpp that can be stimulated by the DksA protein both in vivo and in vitro. Therefore, DksA role could be not only independent but antagonistic to ppGpp in transcription regulation.

The 5'-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex

Xu, N., Gkountela, S., Saeed, K., Akusjarvi, G. — Tue, 15 Sep 2009 07:01:55 PDT

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.

The Gene Wiki: community intelligence applied to human gene annotation

Tue, 15 Sep 2009 06:27:21 PDT

Annotating the function of all human genes is a critical, yet formidable, challenge. Current gene annotation efforts focus on centralized curation resources, but it is increasingly clear that this approach does not scale with the rapid growth of the biomedical literature. The Gene Wiki utilizes an alternative and complementary model based on the principle of community intelligence. Directly integrated within the online encyclopedia, Wikipedia, the goal of this effort is to build a gene-specific review article for every gene in the human genome, where each article is collaboratively written, continuously updated and community reviewed. Previously, we described the creation of Gene Wiki ‘stubs’ for approximately 9000 human genes. Here, we describe ongoing systematic improvements to these articles to increase their utility. Moreover, we retrospectively examine the community usage and improvement of the Gene Wiki, providing evidence of a critical mass of users and editors. Gene Wiki articles are freely accessible within the Wikipedia web site, and additional links and information are available at http://en.wikipedia.org/wiki/Portal:Gene_Wiki.

Recognition of tRNAGln by Helicobacter pylori GluRS2--a tRNAGln-specific glutamyl-tRNA synthetase

Chang, K.-M., Hendrickson, T. L. — Tue, 15 Sep 2009 06:27:18 PDT

Accurate aminoacylation of tRNAs by the aminoacyl-tRNA synthetases (aaRSs) plays a critical role in protein translation. However, some of the aaRSs are missing in many microorganisms. Helicobacter pylori does not have a glutaminyl-tRNA synthetase (GlnRS) but has two divergent glutamyl-tRNA synthetases: GluRS1 and GluRS2. Like a canonical GluRS, GluRS1 aminoacylates tRNA^Glu1 and tRNA^Glu2. In contrast, GluRS2 only misacylates tRNA^Gln to form Glu-tRNA^Gln. It is not clear how GluRS2 achieves specific recognition of tRNA^Gln while rejecting the two H. pylori tRNA^Glu isoacceptors. Here, we show that GluRS2 recognizes major identity elements clustered in the tRNA^Gln acceptor stem. Mutations in the tRNA anticodon or at the discriminator base had little to no impact on enzyme specificity and activity.

Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53

Tue, 15 Sep 2009 06:27:14 PDT

Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K_d of 1.95 ± 0.19 µM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM–p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices 1 and 2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM–p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM–p53 interaction are discussed.

PiggyBac transgenic strategies in the developing chicken spinal cord

Lu, Y., Lin, C., Wang, X. — Tue, 15 Sep 2009 06:27:09 PDT

The chicken spinal cord is an excellent model for the study of early neural development in vertebrates. However, the lack of robust, stable and versatile transgenic methods has limited the usefulness of chick embryos for the study of later neurodevelopmental events. Here we describe a new transgenic approach utilizing the PiggyBac (PB) transposon to facilitate analysis of late-stage neural development such as axon targeting and synaptic connection in the chicken embryo. Using PB transgenic approaches we achieved temporal and spatial regulation of transgene expression and performed stable RNA interference (RNAi). With these new capabilities, we mapped axon projection patterns of V2b subset of spinal interneurons and visualized maturation of the neuromuscular junction (NMJ). Furthermore, PB-mediated RNAi in the chick recapitulated the phenotype of loss of agrin function in the mouse NMJ. The simplicity and versatility of PB-mediated transgenic strategies hold great promise for large-scale genetic analysis of neuronal connectivity in the chick.

Translation of the FMR1 mRNA is not influenced by AGG interruptions

Mon, 14 Sep 2009 07:31:18 PDT

The fragile X mental retardation 1 (FMR1) gene contains a CGG-repeat element within its 5' untranslated region (5'UTR) which, for alleles with more than ~40 repeats, increasingly affects both transcription (up-regulation) and translation (inhibition) of the repeat-containing RNA with increasing CGG-repeat length. Translational inhibition is thought to be due to impaired ribosomal scanning through the CGG-repeat region, which is postulated to form highly stable secondary/tertiary structure. One striking difference between alleles in the premutation range (55–200 CGG repeats) and those in the normal range (<~40 repeats) is the reduced number/absence of ‘expansion stabilizing’ AGG interruptions in the larger alleles. Such interruptions, which generally occur every 9–11 repeats in normal alleles, are thought to disrupt the extended CGG-repeat hairpin structure, thus facilitating translational initiation. To test this hypothesis, we have measured the translational efficiency of CGG-repeat mRNAs with 0–2 AGG interruptions, both in vitro (rabbit reticulocyte lysates) and in cell culture (HEK-293 cells). We demonstrate that the AGG interruptions have no detectable influence on translational efficiency in either a cell-free system or cell culture, indicating that any AGG-repeat-induced alterations in secondary/tertiary structure, if present, do not involve the rate-limiting step(s) in translational initiation.

Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption

Thu, 10 Sep 2009 08:30:08 PDT

The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.

Genome-wide analysis of a G-quadruplex-specific single-chain antibody that regulates gene expression

Thu, 10 Sep 2009 08:29:51 PDT

G-quadruplex nucleic acids have been proposed to play a role in a number of fundamental biological processes that include transcription and translation. We have developed a single-chain antibody that is selective for G-quadruplex DNA over double-stranded DNA, and here show that when it is expressed in human cells, it significantly affects the expression of a wide variety of genes, in a manner that correlates with the presence of predicted G-quadruplexes. We observe cases where gene expression is increased or decreased, and that there are apparent interactions with G-quadruplex motifs at the beginning and end of the genes, and on either strand. The outcomes of this genome-wide study demonstrate that G-quadruplex recognition by the antibody has physiological consequences, and provides insights into some of the complexity associated with G-quadruplex-based regulation.

MeCP2/H3meK9 are involved in IL-6 gene silencing in pancreatic adenocarcinoma cell lines

Dandrea, M., Donadelli, M., Costanzo, C., Scarpa, A., Palmieri, M. — Thu, 10 Sep 2009 08:29:31 PDT

The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2'-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions –666 to –426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2.

Selection for minimization of translational frameshifting errors as a factor in the evolution of codon usage

Huang, Y., Koonin, E. V., Lipman, D. J., Przytycka, T. M. — Thu, 10 Sep 2009 08:29:10 PDT

In a wide range of genomes, it was observed that the usage of synonymous codons is biased toward specific codons and codon patterns. Factors that are implicated in the selection for codon usage include facilitation of fast and accurate translation. There are two types of translational errors: missense errors and processivity errors. There is considerable evidence in support of the hypothesis that codon usage is optimized to minimize missense errors. In contrast, little is known about the relationship between codon usage and frameshifting errors, an important form of processivity errors, which appear to occur at frequencies comparable to the frequencies of missense errors. Based on the recently proposed pause-and-slip model of frameshifting, we developed Frameshifting Robustness Score (FRS). We used this measure to test if the pattern of codon usage indicates optimization against frameshifting errors. We found that the FRS values of protein-coding sequences from four analyzed genomes (the bacteria Bacillus subtilis and Escherichia coli, and the yeasts Saccharomyces cerevisiae and Schizosaccharomyce pombe) were typically higher than expected by chance. Other properties of FRS patterns observed in B. subtilis, S. cerevisiae and S. pombe, such as the tendency of FRS to increase from the 5'- to 3'-end of protein-coding sequences, were also consistent with the hypothesis of optimization against frameshifting errors in translation. For E. coli, the results of different tests were less consistent, suggestive of a much weaker optimization, if any. Collectively, the results fit the concept of selection against mistranslation-induced protein misfolding being one of the factors shaping the evolution of both coding and non-coding sequences.

Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides

Gibson, D. G. — Thu, 10 Sep 2009 08:28:43 PDT

Here it is demonstrated that the yeast Saccharomyces cerevisiae can take up and assemble at least 38 overlapping single-stranded oligonucleotides and a linear double-stranded vector in one transformation event. These oligonucleotides can overlap by as few as 20 bp, and can be as long as 200 nucleotides in length. This straightforward scheme for assembling chemically-synthesized oligonucleotides could be a useful tool for building synthetic DNA molecules.

Regulation of transcription termination in the nematode Caenorhabditis elegans

Wed, 09 Sep 2009 06:53:24 PDT

The current predicted mechanisms that describe RNA polymerase II (pol II) transcription termination downstream of protein expressing genes fail to adequately explain, how premature termination is prevented in eukaryotes that possess operon-like structures. Here we address this issue by analysing transcription termination at the end of single protein expressing genes and genes located within operons in the nematode Caenorhabditis elegans. By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites into the 3' flanking regions of the nematode genes before it terminates. We also show that pol II does not terminate after transcription of internal poly(A) sites in operons. We provide experimental evidence that five randomly chosen C. elegans operons are transcribed as polycistronic pre-mRNAs. Furthermore, we show that cis-splicing of the first intron located in downstream positioned genes in these polycistronic pre-mRNAs is critical for their expression and may play a role in preventing premature pol II transcription termination.

An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

Wed, 09 Sep 2009 06:53:21 PDT

We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a ~21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.

Characterization of RNA aptamers that disrupt the RUNX1-CBF{beta}/DNA complex

Wed, 09 Sep 2009 06:53:18 PDT

The transcription factor RUNX1 (AML1) is an important regulator of haematopoiesis, and an important fusion partner in leukaemic translocations. High-affinity DNA binding by RUNX1 requires the interaction of the RUNX1 Runt-Homology-Domain (RHD) with the core-binding factor β protein (CBFβ). To generate novel reagents for in vitro and in vivo studies of RUNX1 function, we have selected high-affinity RNA aptamers against a recombinant RHD–CBFβ complex. Selection yielded two sequence families, each dominated by a single consensus sequence. Aptamers from each family disrupt DNA binding by the RUNX1 protein in vitro and compete with sequence-specific dsDNA binding. Minimal, high-affinity (~100–160 nM) active aptamer fragments 28 and 30 nts in length, consisting of simple short stem-loop structures, were then identified. These bind to the RHD subunit and disrupt its interaction with CBFβ, which is consistent with reduced DNA affinity in the presence of aptamer. These aptamers represent new reagents that target a novel surface on the RHD required to stabilize the recombinant RHD–CBFβ complex and thus will further aid exploring the functions of this key transcription factor.

In vivo methylation of mtDNA reveals the dynamics of protein-mtDNA interactions

Rebelo, A. P., Williams, S. L., Moraes, C. T. — Wed, 09 Sep 2009 06:53:15 PDT

To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNA^Leu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins.

Predictable suppression of gene expression by 5'-UTR-based RNA quadruplexes

Halder, K., Wieland, M., Hartig, J. S. — Wed, 09 Sep 2009 06:53:12 PDT

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure–function relationship of synthetic quadruplex sequences in 5'-UTRs in multiple mammalian cell-lines. We establish a universal ‘translational suppressor’ effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of ‘GGG’-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.

Recognition and coupling of A-to-I edited sites are determined by the tertiary structure of the RNA

Enstero, M., Daniel, C., Wahlstedt, H., Major, F., Ohman, M. — Tue, 08 Sep 2009 23:07:19 PDT

Adenosine-to-inosine (A-to-I) editing has been shown to be an important mechanism that increases protein diversity in the brain of organisms from human to fly. The family of ADAR enzymes converts some adenosines of RNA duplexes to inosines through hydrolytic deamination. The adenosine recognition mechanism is still largely unknown. Here, to investigate it, we analyzed a set of selectively edited substrates with a cluster of edited sites. We used a large set of individual transcripts sequenced by the 454 sequencing technique. On average, we analyzed 570 single transcripts per edited region at four different developmental stages from embryogenesis to adulthood. To our knowledge, this is the first time, large-scale sequencing has been used to determine synchronous editing events. We demonstrate that edited sites are only coupled within specific distances from each other. Furthermore, our results show that the coupled sites of editing are positioned on the same side of a helix, indicating that the three-dimensional structure is key in ADAR enzyme substrate recognition. Finally, we propose that editing by the ADAR enzymes is initiated by their attraction to one principal site in the substrate.

Evidence for large diversity in the human transcriptome created by Alu RNA editing

Tue, 08 Sep 2009 23:35:00 PDT

Adenosine-to-inosine (A-to-I) RNA editing alters the original genomic content of the human transcriptome and is essential for maintenance of normal life in mammals. A-to-I editing in Alu repeats is abundant in the human genome, with many thousands of expressed Alu sequences undergoing editing. Little is known so far about the contribution of Alu editing to transcriptome complexity. Transcripts derived from a single edited Alu sequence can be edited in multiple sites, and thus could theoretically generate a large number of different transcripts. Here we explored whether the combinatorial potential nature of edited Alu sequences is actually fulfilled in the human transcriptome. We analyzed datasets of editing sites and performed an analysis of a detailed transcript set of one edited Alu sequence. We found that editing appears at many more sites than detected by earlier genomic screens. To a large extent, editing of different sites within the same transcript is only weakly correlated. Thus, rather than finding a few versions of each transcript, a large number of edited variants arise, resulting in immense transcript diversity that eclipses alternative splicing as mechanism of transcriptome diversity, although with less impact on the proteome.

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

Tue, 08 Sep 2009 23:34:54 PDT

Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions.

GSK3{beta} is a negative regulator of the transcriptional coactivator MAML1

Tue, 08 Sep 2009 23:07:16 PDT

Glycogen synthase kinase 3β (GSK3β) is involved in several cellular signaling systems through regulation of the activity of diverse transcription factors such as Notch, p53 and β-catenin. Mastermind-like 1 (MAML1) was originally identified as a Notch coactivator, but has also been reported to function as a transcriptional coregulator of p53, β-catenin and MEF2C. In this report, we show that active GSK3β directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity, suggesting that GSK3β might target a coactivator in its regulation of gene expression. We have previously shown that MAML1 increases global acetylation of histones, and here we show that the GSK3 inhibitor SB41, further enhances MAML1-dependent histone acetylation in cells. Finally, MAML1 translocates GSK3β to nuclear bodies; this function requires full-length MAML1 protein.

The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

Tue, 08 Sep 2009 23:07:13 PDT

In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently.

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Neely, R. K., Tamulaitis, G., Chen, K., Kubala, M., Siksnys, V., Jones, A. C. — Tue, 08 Sep 2009 23:07:08 PDT

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

HMMCONVERTER 1.0: a toolbox for hidden Markov models

Lam, T. Y., Meyer, I. M. — Tue, 08 Sep 2009 23:07:03 PDT

Hidden Markov models (HMMs) and their variants are widely used in Bioinformatics applications that analyze and compare biological sequences. Designing a novel application requires the insight of a human expert to define the model's architecture. The implementation of prediction algorithms and algorithms to train the model's parameters, however, can be a time-consuming and error-prone task. We here present HMMConverter, a software package for setting up probabilistic HMMs, pair-HMMs as well as generalized HMMs and pair-HMMs. The user defines the model itself and the algorithms to be used via an XML file which is then directly translated into efficient C++ code. The software package provides linear-memory prediction algorithms, such as the Hirschberg algorithm, banding and the integration of prior probabilities and is the first to present computationally efficient linear-memory algorithms for automatic parameter training. Users of HMMConverter can thus set up complex applications with a minimum of effort and also perform parameter training and data analyses for large data sets.

External oligonucleotide standards enable cross laboratory comparison and exchange of real-time quantitative PCR data

Fri, 04 Sep 2009 09:18:38 PDT

The quantitative polymerase chain reaction (qPCR) is widely utilized for gene expression analysis. However, the lack of robust strategies for cross laboratory data comparison hinders the ability to collaborate or perform large multicentre studies conducted at different sites. In this study we introduced and validated a workflow that employs universally applicable, quantifiable external oligonucleotide standards to address this question. Using the proposed standards and data-analysis procedure, we obtained a perfect concordance between expression values from eight different genes in 366 patient samples measured on three different qPCR instruments and matching software, reagents, plates and seals, demonstrating the power of this strategy to detect and correct inter-run variation and to enable exchange of data between different laboratories, even when not using the same qPCR platform.

A high throughput experimental approach to identify miRNA targets in human cells

Fri, 04 Sep 2009 09:18:33 PDT

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation—gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that ~40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.

A high-resolution magnetic tweezer for single-molecule measurements

Kim, K., Saleh, O. A. — Thu, 03 Sep 2009 08:49:11 PDT

Magnetic tweezers (MT) are single-molecule manipulation instruments that utilize a magnetic field to apply force to a biomolecule-tethered magnetic bead while using optical bead tracking to measure the biomolecule’s extension. While relatively simple to set up, prior MT implementations have lacked the resolution necessary to observe sub-nanometer biomolecular configuration changes. Here, we demonstrate a reflection-interference technique for bead tracking, and show that it has much better resolution than traditional diffraction-based systems. We enhance the resolution by fabricating optical coatings on all reflecting surfaces that optimize the intensity and contrast of the interference image, and we implement feedback control of the focal position to remove drift. To test the system, we measure the length change of a DNA hairpin as it undergoes a folding/unfolding transition.

A generalized conformational energy function of DNA derived from molecular dynamics simulations

Yamasaki, S., Terada, T., Shimizu, K., Kono, H., Sarai, A. — Thu, 03 Sep 2009 08:49:08 PDT

Proteins recognize DNA sequences by two different mechanisms. The first is direct readout, in which recognition is mediated by direct interactions between the protein and the DNA bases. The second is indirect readout, which is caused by the dependence of conformation and the deformability of the DNA structure on the sequence. Various energy functions have been proposed to evaluate the contribution of indirect readout to the free-energy changes in complex formations. We developed a new generalized energy function to estimate the dependence of the deformability of DNA on the sequence. This function was derived from molecular dynamics simulations previously conducted on B-DNA dodecamers, each of which had one possible tetramer sequence embedded at its center. By taking the logarithm of the probability distribution function (PDF) for the base-step parameters of the central base-pair step of the tetramer, its ability to distinguish the native sequence from random ones was superior to that with the previous method that approximated the energy function in harmonic form. From a comparison of the energy profiles calculated with these two methods, we found that the harmonic approximation caused significant errors in the conformational energies of the tetramers that adopted multiple stable conformations.

Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production

Thu, 03 Sep 2009 08:49:01 PDT

In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.

In vivo expression and purification of aptamer-tagged small RNA regulators

Said, N., Rieder, R., Hurwitz, R., Deckert, J., Urlaub, H., Vogel, J. — Wed, 02 Sep 2009 08:27:59 PDT

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria.

Real-time DNA microarray analysis

Hassibi, A., Vikalo, H., Riechmann, J. L., Hassibi, B. — Mon, 31 Aug 2009 22:48:19 PDT

We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

The fragment structure of a putative HsdR subunit of a type I restriction enzyme from Vibrio vulnificus YJ016: implications for DNA restriction and translocation activity

Uyen, N. T., Park, S.-Y., Choi, J.-W., Lee, H.-J., Nishi, K., Kim, J.-S. — Wed, 22 Jul 2009 09:34:40 PDT

Among four types of bacterial restriction enzymes that cleave a foreign DNA depending on its methylation status, type I enzymes composed of three subunits are interesting because of their unique DNA cleavage and translocation mechanisms performed by the restriction subunit (HsdR). The elucidated N-terminal fragment structure of a putative HsdR subunit from Vibrio vulnificus YJ016 reveals three globular domains. The nucleolytic core within an N-terminal nuclease domain (NTD) is composed of one basic and three acidic residues, which include a metal-binding site. An ATP hydrolase (ATPase) site at the interface of two RecA-like domains (RDs) is located close to the probable DNA-binding site for translocation, which is far from the NTD nucleolytic core. Comparison of relative domain arrangements with other functionally related ATP and/or DNA complex structures suggests a possible translocation and restriction mechanism of the HsdR subunit. Furthermore, careful analysis of its sequence and structure implies that a linker helix connecting two RDs and an extended region within the nuclease domain may play a central role in switching the DNA translocation into the restriction activity.

Specific gene silencing by artificial trans-encoded small noncoding RNAs in bacteria

Cheng, R., Miao, C., Gong, Q., Gu, Y., Lu, X., Han, F., Yu, W. — Wed, 27 May 2009 10:12:40 PDT

Recently, numerous small noncoding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs, acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become an extraordinarily powerful RNA silencing tool for elucidating and manipulating gene functions in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes. In this study, we described firstly the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of natural bacterial trans-encoded sRNAs, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base-pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. atsRNA led to translational repression and RNase-E-dependent degradation of target mRNA, and the translation inhibition was the primary event for gene silencing. Our findings demonstrated that atsRNA was an effective RNA tool for specific gene silencing in bacteria.

This paper has been withdrawn

Tue, 12 Jun 2007 01:36:08 PDT