Roland Arnold

MIPS: analysis and annotation of proteins from whole genomes

The Munich Information Center for Protein Sequences (MIPS at the GSF), Neuherberg, Germany, provides resources related to genome information. Manually curated databases for several reference organisms are maintained. Several of these databases are described elsewhere in this and other recent NAR database issues. In a complementary effort, a comprehensive set of >400 genomes automatically annotated with the PEDANT system are maintained. The main goal of our current work on creating and maintaining genome databases is to extend gene centered information to information on interactions within a generic comprehensive framework. We have concentrated our efforts along three lines (i) the development of suitable comprehensive data structures and database technology, communication and query tools to include a wide range of different types of information enabling the representation of complex information such as functional modules or networks Genome Research Environment System, (ii) the development of databases covering computable information such as the basic evolutionary relations among all genes, namely SIMAP, the sequence similarity matrix and the CABiNet network analysis framework and (iii) the compilation and manual annotation of information related to interactions such as protein-protein interactions or other types of relations (e.g. MPCDB, MPPI, CYGD). All databases described and the detailed descriptions of our projects can be accessed through the MIPS WWW server (http://mips.gsf.de).

eggNOG v3.0: orthologous groups covering 1133 organisms at 41 different taxonomic ranges

Orthologous relationships form the basis of most comparative genomic and metagenomic studies and are essential for proper phylogenetic and functional analyses. The third version of the eggNOG database (http://eggnog.embl.de) contains non-supervised orthologous groups constructed from 1133 organisms, doubling the number of genes with orthology assignment compared to eggNOG v2. The new release is the result of a number of improvements and expansions: (i) the underlying homology searches are now based on the SIMAP database; (ii) the orthologous groups have been extended to 41 levels of selected taxonomic ranges enabling much more fine-grained orthology assignments; and (iii) the newly designed web page is considerably faster with more functionality. In total, eggNOG v3 contains 721 801 orthologous groups, encompassing a total of 4 396 591 genes. Additionally, we updated 4873 and 4850 original COGs and KOGs, respectively, to include all 1133 organisms. At the universal level, covering all three domains of life, 101 208 orthologous groups are available, while the others are applicable at 40 more limited taxonomic ranges. Each group is amended by multiple sequence alignments and maximum-likelihood trees and broad functional descriptions are provided for 450 904 orthologous groups (62.5%).

Gepard: a rapid and sensitive tool for creating dotplots on genome scale

UNLABELLED Gepard provides a user-friendly, interactive application for the quick creation of dotplots. It utilizes suffix arrays to reduce the time complexity of dotplot calculation to Theta(m*log n). A client-server mode, which is a novel feature for dotplot creation software, allows the user to calculate dotplots and color them by functional annotation without any prior downloading of sequence or annotation data. AVAILABILITY Both source codes and executable binaries are available at http://mips.gsf.de/services/analysis/gepard

MIPS Arabidopsis thaliana Database (MAtDB): an integrated biological knowledge resource based on the first complete plant genome

Arabidopsis thaliana is the first plant for which the complete genome has been sequenced and published. Annotation of complex eukaryotic genomes requires more than the assignment of genetic elements to the sequence. Besides completing the list of genes, we need to discover their cellular roles, their regulation and their interactions in order to understand the workings of the whole plant. The MIPS Arabidopsis thaliana Database (MAtDB; http://mips.gsf.de/proj/thal/db) started out as a repository for genome sequence data in the European Scientists Sequencing Arabidopsis (ESSA) project and the Arabidopsis Genome Initiative. Our aim is to transform MAtDB into an integrated biological knowledge resource by integrating diverse data, tools, query and visualization capabilities and by creating a comprehensive resource for Arabidopsis as a reference model for other species, including crop plants.

Sequence-Based Prediction of Type III Secreted Proteins

The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of ∼71% and selectivity of ∼85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will facilitate further studies on and improve our understanding of type III secretion and its role in pathogen–host interactions.

probeCheck – a central resource for evaluating oligonucleotide probe coverage and specificity

The web server probeCheck, freely accessible at http://www.microbial-ecology.net/probecheck, provides a pivotal forum for rapid specificity and coverage evaluations of probes and primers against selected databases of phylogenetic and functional marker genes. Currently, 24 widely used sequence collections including the Ribosomal Database Project (RDP) II, Greengenes, SILVA and the Functional Gene Pipeline/Repository can be queried. For this purpose, probeCheck integrates a new online version of the popular ARB probe match tool with free energy (ΔG) calculations for each perfectly matched and mismatched probe-target hybrid, allowing assessment of the theoretical binding stabilities of oligo-target and non-target hybrids. For each output sequence, the accession number, the GenBank taxonomy and a link to the respective entry at GenBank, EMBL and, if applicable, the query database are displayed. Filtering options allow customizing results on the output page. In addition, probeCheck is linked with probe match tools of RDP II and Greengenes, NCBI blast, the Oligonucleotide Properties Calculator, the two-state folding tool of the DINAMelt server and the rRNA-targeted probe database probeBase. Taken together, these features provide a multifunctional platform with maximal flexibility for the user in the choice of databases and options for the evaluation of published and newly developed probes and primers.

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

DNA replication−associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10−13). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

B2G-FAR, a species-centered GO annotation repository

Motivation: Functional genomics research has expanded enormously in the last decade thanks to the cost reduction in high-throughput technologies and the development of computational tools that generate, standardize and share information on gene and protein function such as the Gene Ontology (GO). Nevertheless, many biologists, especially working with non-model organisms, still suffer from non-existing or low-coverage functional annotation, or simply struggle retrieving, summarizing and querying these data. Results: The Blast2GO Functional Annotation Repository (B2G-FAR) is a bioinformatics resource envisaged to provide functional information for otherwise uncharacterized sequence data and offers data mining tools to analyze a larger repertoire of species than currently available. This new annotation resource has been created by applying the Blast2GO functional annotation engine in a strongly high-throughput manner to the entire space of public available sequences. The resulting repository contains GO term predictions for over 13.2 million non-redundant protein sequences based on BLAST search alignments from the SIMAP database. We generated GO annotation for approximately 150 000 different taxa making available 2000 species with the highest coverage through B2G-FAR. A second section within B2G-FAR holds functional annotations for 17 non-model organism Affymetrix GeneChips. Conclusions: B2G-FAR provides easy access to exhaustive functional annotation for 2000 species offering a good balance between quality and quantity, thereby supporting functional genomics research especially in the case of non-model organisms. Availability: The annotation resource is available at http://www.b2gfar.org. Contact: aconesa@cipf.es; sgoetz@cipf.es Supplementary information: Supplementary data are available at Bioinformatics online.

The binary protein-protein interaction landscape of Escherichia coli

Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (∼70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, which approximately doubles the number of known binary PPIs in E. coli. Integration of binary PPI and genetic-interaction data revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that we could map in multiprotein complexes were informative regarding internal topology of complexes and indicated that interactions in complexes are substantially more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily important model microbe.

PEDANT covers all complete RefSeq genomes

The PEDANT genome database provides exhaustive annotation of nearly 3000 publicly available eukaryotic, eubacterial, archaeal and viral genomes with more than 4.5 million proteins by a broad set of bioinformatics algorithms. In particular, all completely sequenced genomes from the NCBIs Reference Sequence collection (RefSeq) are covered. The PEDANT processing pipeline has been sped up by an order of magnitude through the utilization of precalculated similarity information stored in the similarity matrix of proteins (SIMAP) database, making it possible to process newly sequenced genomes immediately as they become available. PEDANT is freely accessible to academic users at http://pedant.gsf.de. For programmatic access Web Services are available at http://pedant.gsf.de/webservices.jsp.