Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies target genes for male infertility.

BACKGROUND: Mammalian spermatogenesis is a process that involves a complex expression program in both somatic and germ cells present in the male gonad. A number of studies have attempted to define the transcriptome of male meiosis and gametogenesis in rodents and primates. Few human transcripts, however, have been associated with testicular somatic cells and germ cells at different post-natal developmental stages and little is known about their level of germline-specificity compared with non-testicular tissues. METHODS: We quantified human transcripts using GeneChips and a total of 47 biopsies from prepubertal children diagnosed with undescended testis, infertile adult patients whose spermatogenesis is arrested at consecutive stages and fertile control individuals. These results were integrated with data from enriched normal germ cells, non-testicular expression data, phenotype information, predicted regulatory DNA-binding motifs and interactome data. RESULTS: Among 3580 genes for which we found differential transcript concentrations in somatic and germ cells present in human testis, 933 were undetectable in 45 embryonic and adult non-testicular tissues, including many that were corroborated at protein level by published gene annotation data and histological high-throughput protein immunodetection assays. Using motif enrichment analyses, we identified regulatory promoter elements likely involved in germline development. Finally, we constructed a regulatory disease network for human fertility by integrating expression signals, interactome information, phenotypes and functional annotation data. CONCLUSIONS: Our results provide broad insight into the post-natal human testicular transcriptome at the level of cell populations and in a global somatic tissular context. Furthermore, they yield clues for genetic causes of male infertility and will facilitate the identification of novel cancer/testis genes as targets for cancer immunotherapies.

The Microbe browser for comparative genomics.

The Microbe browser is a web server providing comparative microbial genomics data. It offers comprehensive, integrated data from GenBank, RefSeq, UniProt, InterPro, Gene Ontology and the Orthologs Matrix Project (OMA) database, displayed along with gene predictions from five software packages. The Microbe browser is daily updated from the source databases and includes all completely sequenced bacterial and archaeal genomes. The data are displayed in an easy-to-use, interactive website based on Ensembl software. The Microbe browser is available at http://microbe.vital-it.ch/. Programmatic access is available through the OMA application programming interface (API) at http://microbe.vital-it.ch/api.

MIMAS 3.0 is a Multiomics Information Management and Annotation System.

BACKGROUND: DNA sequence integrity, mRNA concentrations and protein-DNA interactions have been subject to genome-wide analyses based on microarrays with ever increasing efficiency and reliability over the past fifteen years. However, very recently novel technologies for Ultra High-Throughput DNA Sequencing (UHTS) have been harnessed to study these phenomena with unprecedented precision. As a consequence, the extensive bioinformatics environment available for array data management, analysis, interpretation and publication must be extended to include these novel sequencing data types. DESCRIPTION: MIMAS was originally conceived as a simple, convenient and local Microarray Information Management and Annotation System focused on GeneChips for expression profiling studies. MIMAS 3.0 enables users to manage data from high-density oligonucleotide SNP Chips, expression arrays (both 3'UTR and tiling) and promoter arrays, BeadArrays as well as UHTS data using MIAME-compliant standardized vocabulary. Importantly, researchers can export data in MAGE-TAB format and upload them to the EBI's ArrayExpress certified data repository using a one-step procedure. CONCLUSION: We have vastly extended the capability of the system such that it processes the data output of six types of GeneChips (Affymetrix), two different BeadArrays for mRNA and miRNA (Illumina) and the Genome Analyzer (a popular Ultra-High Throughput DNA Sequencer, Illumina), without compromising on its flexibility and user-friendliness. MIMAS, appropriately renamed into Multiomics Information Management and Annotation System, is currently used by scientists working in approximately 50 academic laboratories and genomics platforms in Switzerland and France. MIMAS 3.0 is freely available via http://multiomics.sourceforge.net/.

The GermOnline cross-species systems browser provides comprehensive information on genes and gene products relevant for sexual reproduction.

We report a novel release of the GermOnline knowledgebase covering genes relevant for the cell cycle, gametogenesis and fertility. GermOnline was extended into a cross-species systems browser including information on DNA sequence annotation, gene expression and the function of gene products. The database covers eight model organisms and Homo sapiens, for which complete genome annotation data are available. The database is now built around a sophisticated genome browser (Ensembl), our own microarray information management and annotation system (MIMAS) used to extensively describe experimental data obtained with high-density oligonucleotide microarrays (GeneChips) and a comprehensive system for online editing of database entries (MediaWiki). The RNA data include results from classical microarrays as well as tiling arrays that yield information on RNA expression levels, transcript start sites and lengths as well as exon composition. Members of the research community are solicited to help GermOnline curators keep database entries on genes and gene products complete and accurate. The database is accessible at http://www.germonline.org/.

ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins.

ScanProsite--http://www.expasy.org/tools/scanprosite/--is a new and improved version of the web-based tool for detecting PROSITE signature matches in protein sequences. For a number of PROSITE profiles, the tool now makes use of ProRules--context-dependent annotation templates--to detect functional and structural intra-domain residues. The detection of those features enhances the power of function prediction based on profiles. Both user-defined sequences and sequences from the UniProt Knowledgebase can be matched against custom patterns, or against PROSITE signatures. To improve response times, matches of sequences from UniProtKB against PROSITE signatures are now retrieved from a pre-computed match database. Several output modes are available including simple text views and a rich mode providing an interactive match and feature viewer with a graphical representation of results.

Ashbya Genome Database 3.0: a cross-species genome and transcriptome browser for yeast biologists.

BACKGROUND: The Ashbya Genome Database (AGD) 3.0 is an innovative cross-species genome and transcriptome browser based on release 40 of the Ensembl developer environment. DESCRIPTION: AGD 3.0 provides information on 4726 protein-encoding loci and 293 non-coding RNA genes present in the genome of the filamentous fungus Ashbya gossypii. A synteny viewer depicts the chromosomal location and orientation of orthologous genes in the budding yeast Saccharomyces cerevisiae. Genome-wide expression profiling data obtained with high-density oligonucleotide microarrays (GeneChips) are available for nearly all currently annotated protein-coding loci in A. gossypii and S. cerevisiae. CONCLUSION: AGD 3.0 hence provides yeast- and genome biologists with comprehensive report pages including reliable DNA annotation, Gene Ontology terms associated with S. cerevisiae orthologues and RNA expression data as well as numerous links to external sources of information. The database is accessible at http://agd.vital-it.ch/.

Integr8 and Genome Reviews: integrated views of complete genomes and proteomes.

Integr8 is a new web portal for exploring the biology of organisms with completely deciphered genomes. For over 190 species, Integr8 provides access to general information, recent publications, and a detailed statistical overview of the genome and proteome of the organism. The preparation of this analysis is supported through Genome Reviews, a new database of bacterial and archaeal DNA sequences in which annotation has been upgraded (compared to the original submission) through the integration of data from many sources, including the EMBL Nucleotide Sequence Database, the UniProt Knowledgebase, InterPro, CluSTr, GOA and HOGENOM. Integr8 also allows the users to customize their own interactive analysis, and to download both customized and prepared datasets for their own use. Integr8 is available at http://www.ebi.ac.uk/integr8.

ExPASy: The proteomics server for in-depth protein knowledge and analysis.

The ExPASy (the Expert Protein Analysis System) World Wide Web server (http://www.expasy.org), is provided as a service to the life science community by a multidisciplinary team at the Swiss Institute of Bioinformatics (SIB). It provides access to a variety of databases and analytical tools dedicated to proteins and proteomics. ExPASy databases include SWISS-PROT and TrEMBL, SWISS-2DPAGE, PROSITE, ENZYME and the SWISS-MODEL repository. Analysis tools are available for specific tasks relevant to proteomics, similarity searches, pattern and profile searches, post-translational modification prediction, topology prediction, primary, secondary and tertiary structure analysis and sequence alignment. These databases and tools are tightly interlinked: a special emphasis is placed on integration of database entries with related resources developed at the SIB and elsewhere, and the proteomics tools have been designed to read the annotations in SWISS-PROT in order to enhance their predictions. ExPASy started to operate in 1993, as the first WWW server in the field of life sciences. In addition to the main site in Switzerland, seven mirror sites in different continents currently serve the user community.

Recent improvements to the PROSITE database.

The PROSITE database consists of a large collection of biologically meaningful signatures that are described as patterns or profiles. Each signature is linked to documentation that provides useful biological information on the protein family, domain or functional site identified by the signature. The PROSITE web page has been redesigned and several tools have been implemented to help the user discover new conserved regions in their own proteins and to visualize domain arrangements. We also introduced the facility to search PDB with a PROSITE entry or a user's pattern and visualize matched positions on 3D structures. The latest version of PROSITE (release 18.17 of November 30, 2003) contains 1676 entries. The database is accessible at http://www.expasy.org/prosite/.

The Swiss-Prot variant page and the ModSNP database: a resource for sequence and structure information on human protein variants.

Missense mutation leading to single amino acid polymorphism (SAP) is the type of mutation most frequently related to human diseases. The Swiss-Prot protein knowledgebase records information on such mutations in various sections of a protein entry, namely in the "feature," "comment," and "reference" fields. To facilitate users in obtaining the most relevant information about each human SAP recorded in the knowledgebase, the Swiss-Prot Variant web pages were created to provide a summary of available sequence information, as well as additional structural information on each variant. In particular, the ModSNP database was set up to store information related to SAPs and to manage the modeling of SAPs onto protein structures via an automatic homology modeling pipeline. Currently, among the 16,566 human SAPs recorded in the Swiss-Prot knowledgebase (release 42.5, 21 November 2003), more than 25% have corresponding 3D-models. Of these variants, 47% are related to disease, 26% are polymorphisms, and 27% are not yet clearly classified. The ModSNP database is updated and the subsequent model construction pipeline is launched with each weekly Swiss-Prot release. Thus, the ModSNP database represents a valuable resource for the structural analysis of protein variation. The Swiss-Prot variant pages are accessible from the NiceProt view of a Swiss-Prot entry on the ExPASy server (www.expasy.org/), via a hyperlink created for the stable and unique identifier FTId of each human SAP.

ScanProsite: a reference implementation of a PROSITE scanning tool.

Many different software tools are available publicly to scan the PROSITE database of protein families. However, none of them, to our knowledge, wholly implements the PROSITE syntax, or satisfies all the rules for scanning a pattern against a sequence. We hereby propose a strict definition of how a PROSITE pattern is to be scanned against a sequence, and provide a reference implementation of a tool to scan PROSITE patterns, rules and profiles against protein sequences.

Automated annotation of microbial proteomes in SWISS-PROT.

Large-scale sequencing of prokaryotic genomes demands the automation of certain annotation tasks currently manually performed in the production of the SWISS-PROT protein knowledgebase. The HAMAP project, or 'High-quality Automated and Manual Annotation of microbial Proteomes', aims to integrate manual and automatic annotation methods in order to enhance the speed of the curation process while preserving the quality of the database annotation. Automatic annotation is only applied to entries that belong to manually defined orthologous families and to entries with no identifiable similarities (ORFans). Many checks are enforced in order to prevent the propagation of wrong annotation and to spot problematic cases, which are channelled to manual curation. The results of this annotation are integrated in SWISS-PROT, and a website is provided at http://www.expasy.org/sprot/hamap/.

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

GermOnline 4.0 is a genomics gateway for germline development, meiosis and the mitotic cell cycle.

GermOnline 4.0 is a cross-species database portal focusing on high-throughput expression data relevant for germline development, the meiotic cell cycle and mitosis in healthy versus malignant cells. It is thus a source of information for life scientists as well as clinicians who are interested in gene expression and regulatory networks. The GermOnline gateway provides unlimited access to information produced with high-density oligonucleotide microarrays (3'-UTR GeneChips), genome-wide protein-DNA binding assays and protein-protein interaction studies in the context of Ensembl genome annotation. Samples used to produce high-throughput expression data and to carry out genome-wide in vivo DNA binding assays are annotated via the MIAME-compliant Multiomics Information Management and Annotation System (MIMAS 3.0). Furthermore, the Saccharomyces Genomics Viewer (SGV) was developed and integrated into the gateway. SGV is a visualization tool that outputs genome annotation and DNA-strand specific expression data produced with high-density oligonucleotide tiling microarrays (Sc_tlg GeneChips) which cover the complete budding yeast genome on both DNA strands. It facilitates the interpretation of expression levels and transcript structures determined for various cell types cultured under different growth and differentiation conditions. Database URL: www.germonline.org/

Comparative genomic and phylogeographic analysis of Mycobacterium leprae.

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38x coverage) and NHDP63 (46x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy.

The conserved transcriptome in human and rodent male gametogenesis.

We report a cross-species expression profiling analysis of the human, mouse, and rat male meiotic transcriptional program, using enriched germ cell populations, whole gonads, and high-density oligonucleotide microarrays (GeneChips). Among 35% of the protein-coding genes present in rodent and human genomes that were found to be differentially expressed between germ cells and somatic controls, a key group of 357 conserved core loci was identified that displays highly similar meiotic and postmeiotic patterns of transcriptional induction across all three species. Genes known to be important for sexual reproduction are significantly enriched among differentially expressed core loci and a smaller group of conserved genes not detected in 17 nontesticular somatic tissues, correlating transcriptional activation and essential function in the male germ line. Some genes implicated in the etiology of cancer are found to be strongly transcribed in testis, suggesting that these genes may play unexpected roles in sexual reproduction. Expression profiling data further identified numerous conserved genes of biological and clinical interest previously unassociated with the mammalian male germ line.

FindPept, a tool to identify unmatched masses in peptide mass fingerprinting protein identification.

FindPept (http://www.expasy.org/tools/findpept.html) is a software tool designed to identify the origin of peptide masses obtained by peptide mass fingerprinting which are not matched by existing protein identification tools. It identifies masses resulting from unspecific proteolytic cleavage, missed cleavage, protease autolysis or keratin contaminants. It also takes into account post-translational modifications derived from the annotation of the SWISS-PROT database or supplied by the user, and chemical modifications of peptides. Based on a number of experimental examples, we show that the commonly held rules for the specificity of tryptic cleavage are an oversimplification, mainly because of effects of neighboring residues, experimental conditions, and contaminants present in the enzyme sample.

High-quality protein knowledge resource: SWISS-PROT and TrEMBL.

SWISS-PROT is a curated protein sequence database which strives to provide a high level of annotation (such as the description of the function of a protein, its domain structure, post-translational modifications, variants, etc.), a minimal level of redundancy and a high level of integration with other databases. Together with its automatically annotated supplement TrEMBL, it provides a comprehensive and high-quality view of the current state of knowledge about proteins. Ongoing developments include the further improvement of functional and automatic annotation in the databases including evidence attribution with particular emphasis on the human, archaeal and bacterial proteomes and the provision of additional resources such as the International Protein Index (IPI) and XML format of SWISS-PROT and TrEMBL to the user community.

PROSITE: a documented database using patterns and profiles as motif descriptors.

Among the various databases dedicated to the identification of protein families and domains, PROSITE is the first one created and has continuously evolved since. PROSITE currently consists of a large collection of biologically meaningful motifs that are described as patterns or profiles, and linked to documentation briefly describing the protein family or domain they are designed to detect. The close relationship of PROSITE with the SWISS-PROT protein database allows the evaluation of the sensitivity and specificity of the PROSITE motifs and their periodic reviewing. In return, PROSITE is used to help annotate SWISS-PROT entries. The main characteristics and the techniques of family and domain identification used by PROSITE are reviewed in this paper.