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List of the Tumor types having mutations in both MYH9 and RHOA genes in the cBioPortal for Cancer Genomics database

List of the tumor specific mutations identified by whole-genome sequencing and validated in the 10 pairs of MPM tumor and germline DNA samples

Expression levels of candidate genes in 151 patients by gender and histology

List of SNVs identified in the analyzed genes

Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs more frequently in men, but is associated with longer survival in women. Insight into the survival advantage of female patients may advance the molecular understanding of MPM and identify therapeutic interventions that will improve the prognosis for all MPM patients. In this study, we performed whole-genome sequencing of tumor specimens from 10 MPM patients and matched control samples to identify potential driver mutations underlying MPM. We identified molecular differences associated with gender and histology. Specifically, single-nucleotide variants of BAP1 were observed in 21% of cases, with lower mutation rates observed in sarcomatoid MPM (P < 0.001). Chromosome 22q loss was more frequently associated with the epithelioid than that nonepitheliod histology (P = 0.037), whereas CDKN2A deletions occurred more frequently in nonepithelioid subtypes among men (P = 0.021) and were correlated with shorter overall survival for the entire cohort (P = 0.002) and for men (P = 0.012). Furthermore, women were more likely to harbor TP53 mutations (P = 0.004). Novel mutations were found in genes associated with the integrin-linked kinase pathway, including MYH9 and RHOA. Moreover, expression levels of BAP1, MYH9, and RHOA were significantly higher in nonepithelioid tumors, and were associated with significant reduction in survival of the entire cohort and across gender subgroups. Collectively, our findings indicate that diverse mechanisms highly related to gender and histology appear to drive MPM. Cancer Res; 76(2); 319–28. ©2015 AACR.

List of the 51 canonical pathways significantly enriched (p

Schematic representation of the SNVs and/or chromosomal aberrations identified in BAP1, NF2, TP53, MYH9, RHOA, MYH6, MYH10, PIK3C2A, TNFRSF1A, and 22q and 9p in a panel of 147 MPM tumors.

Expression levels for BAP1, NF2, TP53, MYH9, MYH6, MYH10, PIK3C2A, RHOA, and TNFRSF1A detected by Affymetrix® Human Gene 1.1 ST Array in 151 MPM

Whole genome sequencing data analysis of 10 MPM tumor and matched DNA generated by Complete Genomics platform

Journal of biotechnology 261, 37-45 (2017). doi:10.1016/j.jbiotec.2017.07.006 special issue: "Bioinformatics Solutions for Big Data Analysis in Life Sciences presented by the German Network for Bioinformatics Infrastructure / Edited by Alf Puehler", Published by Elsevier Science, Amsterdam [u.a.]

Aim: Macrophage infiltration contributes to the pathogenesis of Type 2 diabetes. Mesenchymal stem cells (MSCs) possess immunomodulatory properties, making them an ideal candidate for therapeutic intervention. This study investigated whether MSCs can modulate the phenotype of monocytes isolated from Type 2 diabetic patients with end-stage renal disease. Materials & methods: Monocytes from control (n = 4) and Type 2 diabetic patients with end-stage renal disease (n = 5) were assessed using flow cytometry and microarray profiling, following 48 h of co-culture with MSCs. Results: Control subjects had a greater proportion of CD14++CD16- monocytes while diabetic patients had a higher proportion of CD14++CD16+ and CD14+CD16++ monocytes. MSCs promoted the proliferation of monocytes isolated from diabetic patients, reduced HLA-DR expression in both groups and promoted the expression of anti-inflammatory genes. Conclusion: MSC-derived factors alter the polarization of monocytes isolated from healthy and diabetic subjects toward an M2 phenotype.

Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs more frequently in men, but is associated with longer survival in women. Insight into the survival advantage of female patients may advance the molecular understanding of MPM and identify therapeutic interventions that will improve the prognosis for all MPM patients. In this study, we performed whole-genome sequencing of tumor specimens from 10 MPM patients and matched control samples to identify potential driver mutations underlying MPM. We identified molecular differences associated with gender and histology. Specifically, single-nucleotide variants of BAP1 were observed in 21% of cases, with lower mutation rates observed in sarcomatoid MPM (P < 0.001). Chromosome 22q loss was more frequently associated with the epithelioid than that nonepitheliod histology (P = 0.037), whereas CDKN2A deletions occurred more frequently in nonepithelioid subtypes among men (P = 0.021) and were correlated with shorter overall survival for the entire cohort (P = 0.002) and for men (P = 0.012). Furthermore, women were more likely to harbor TP53 mutations (P = 0.004). Novel mutations were found in genes associated with the integrin-linked kinase pathway, including MYH9 and RHOA. Moreover, expression levels of BAP1, MYH9, and RHOA were significantly higher in nonepithelioid tumors, and were associated with significant reduction in survival of the entire cohort and across gender subgroups. Collectively, our findings indicate that diverse mechanisms highly related to gender and histology appear to drive MPM. Cancer Res; 76(2); 319–28. ©2015 AACR.

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.

The identification of the genes that participate at the biological interface of two species remains critical to our understanding of the mechanisms of disease resistance, disease susceptibility and symbiosis. The sequencing of complementary DNA (cDNA) libraries prepared from the biological interface between two organisms provides an inexpensive way to identify the novel genes that may be expressed as a cause or consequence of compatible or incompatible interactions. Sequence classification and annotation of species origin typically use an orthology-based approach and require access to large portions of either genome, or a close relative. Novel species- or clade-specific sequences may have no counterpart within existing databases and remain ambiguous features. Here we present a web-service, Eclair, which utilizes support vector machines for the classification of the origin of expressed sequence tags stemming from mixed host cDNA libraries. In addition to providing an interface for the classification of sequences, users are presented with the opportunity to train a model to suit their preferred species pair. Eclair is freely available at http://eclair.btk.fi.

The subunit compositions of two types of NAD(P)H dehydrogenase complexes of Synechocystis sp. PCC 6803, NDH-1L and NDH-1M, were studied by two-dimensional blue-native/SDS-PAGE followed by electrospray tandem mass spectrometry. Fifteen proteins were observed in NDH-1L including hydrophilic subunits (NdhH, -K, -I, -J, -M, and -N) and hydrophobic subunits (NdhA, -B, -E, -G, -D1, and -F1). In addition, NdhL and a novel subunit, Ssl1690 (designated NdhO), were shown to be components of this complex. All subunits mentioned above were present in the NDH-1M complex except NdhD1 and NdhF1. NdhL and Ssl1690 (NdhO) were homologous to hypothetical proteins encoded by genomic DNA in higher plants, suggesting that chloroplast NDH-1 complexes contain related subunits. Diagnostic sequence motifs were found for both NdhL and NdhO homologous proteins. Analysis of ndhL deletion mutant (M9) revealed the presence of assembled NDH-1L and NDH-1M complexes, but these complexes appear to be functionally impaired in the absence of NdhL. Both NDH-1 complexes were absent in the ndhB deletion mutant (M55).

Molecular markers are required in a broad spectrum of gene screening approaches, ranging from gene-mapping within traditional 'forward'-genetics approaches through QTL identification studies to genotyping and haplotyping studies. As we enter the post-genomics era, the need for genetic markers does not diminish, even in the species with fully sequenced genomes. PlantMarkers is a genetic marker database that contains a comprehensive pool of predicted molecular markers. We have adopted contemporary techniques to identify putative single nucleotide polymorphism (SNP), simple sequence repeat (SSR) and conserved orthologue set markers. A systematic approach to identify as broad a range of putative markers has been undertaken by screening the available openSputnik unigene consensus sequences from over 50 plant species. A web presence at http://markers.btk.fi provides functionality so that a user may search for species-specific markers on the basis of many specific criteria not limited to non-synonymous SNPs segregating between different varieties or measured polymorphic SSRs. Feedback forms are provided with all sequence entries to enable inclusion of, for example, map location for markers validated by the research community.

The public expressed sequence tag collections are continually being enriched with high-quality sequences that represent an ever-expanding range of taxonomically diverse plant species. While these sequence collections provide biased insight into the populations of expressed genes available within individual species and their associated tissues, the information is conceivably of wider relevance in a comparative context. When we consider the available expressed sequence tag (EST) collections of summer 2004, most of the major plant taxonomic clades are at least superficially represented. Investigation of the five million available plant ESTs provides a wealth of information that has applications in modelling the routes of plant genome evolution and the identification of lineage-specific genes and gene families. Over four million ESTs from over 50 distinct plant species have been collated within an EST analysis pipeline called openSputnik. The ESTs were resolved down into approximately one million unigene sequences. These have been annotated using orthology-based annotation transfer from reference plant genomes and using a variety of contemporary bioinformatics methods to assign peptide, structural and functional attributes. The openSputnik database is available at http://sputnik.btk.fi.

To provide resources for barley genomics, 110,981 expressed sequence tags (ESTs) were generated from 22 cDNA libraries representing tissues at various developmental stages. This EST collection corresponds to approximately one-third of the 380,000 publicly available barley ESTs. Clustering and assembly resulted in 14,151 tentative consensi (TCs) and 11 073 singletons, altogether representing 25 224 putatively unique sequences. Of these, 17.5% showed no significant similarity to other barley ESTs present in dbEST. More than 41% of all barley genes are supposed to belong to multigene families and approximately 4% of the barley genes undergo alternative splicing. Based on the functional annotation of the set of unique sequences, the functional category 'Energy' was further analysed to reveal tissue- and stage-specific differences in gene expression. Hierarchical clustering of 362 differentially expressed TCs resulted in the identification of seven major clusters. The clusters reflect biochemical pathways predominantly activated in specific tissues and at various developmental stages. During seed germination glycolysis could be identified as the most predominant biochemical pathway. Germination-specific glycolysis is characterized by the coordinated expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase, whose antagonistic actions possibly regulate the flux of amino acids into protein biosynthesis and gluconeogenesis respectively. The expression of defence-related and antioxidant genes during germination might be controlled by the ethylene-signalling pathway as concluded from the coordinated expression of those genes and the transcription factors (TF) EIN3 and EREBPG. Moreover, because of their predominant expression in germinating seeds, TF of the AP2 and MYB type are presumably major regulators of germination.

We carried out a genome-wide prediction of scaffold/matrix attachment regions (S/MARs) in Arabidopsis. Results indicate no uneven distribution on the chromosomal level but a clear underrepresentation of S/MARs inside genes. In cases where S/MARs were predicted within genes, these intragenic S/MARs were preferentially located within the 5′-half, most prominently within introns 1 and 2. Using Arabidopsis whole-genome expression data generated by the massively parallel signature sequencing methodology, we found a negative correlation between S/MAR-containing genes and transcriptional abundance. Expressed sequence tag data correlated the same way with S/MAR-containing genes. Thus, intragenic S/MARs show a negative correlation with transcription level. For various genes it has been shown experimentally that S/MARs can function as transcriptional regulators and that they have an implication in stabilizing expression levels within transgenic plants. On the basis of a genome-wide in silico S/MAR analysis, we found a significant correlation between the presence of intragenic S/MARs and transcriptional down-regulation.

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes—more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca21 signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.

Helicobacter pylori is the main bacterial causative agent of gastroduodenal disorders and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The draft genomes of 10 closely related H. pylori isolates from the multiracial Malaysian population will provide an insight into the genetic diversity of isolates in Southeast Asia. These isolates were cultured from gastric biopsy samples from patients with functional dyspepsia and gastric cancer. The availability of this genomic information will provide an opportunity for examining the evolution and population structure of H. pylori isolates from Southeast Asia, where the East meets the West.

Background Lichens are symbiotic organisms that have a remarkable ability to survive in some of the most extreme terrestrial climates on earth. Lichens can endure frequent desiccation and wetting cycles and are able to survive in a dehydrated molecular dormant state for decades at a time. Genetic resources have been established in lichen species for the study of molecular systematics and their taxonomic classification. No lichen species have been characterised yet using genomics and the molecular mechanisms underlying the lichen symbiosis and the fundamentals of desiccation tolerance remain undescribed. We report the characterisation of a transcriptome of the grey reindeer lichen, Cladonia rangiferina, using high-throughput next-generation transcriptome sequencing and traditional Sanger EST sequencing data. Results Altogether 243,729 high quality sequence reads were de novo assembled into 16,204 contigs and 49,587 singletons. The genome of origin for the sequences produced was predicted using Eclat with sequences derived from the axenically grown symbiotic partners used as training sequences for the classification model. 62.8% of the sequences were classified as being of fungal origin while the remaining 37.2% were predicted as being of algal origin. The assembled sequences were annotated by BLASTX comparison against a non-redundant protein sequence database with 34.4% of the sequences having a BLAST match. 29.3% of the sequences had a Gene Ontology term match and 27.9% of the sequences had a domain or structural match following an InterPro search. 60 KEGG pathways with more than 10 associated sequences were identified. Conclusions Our results present a first transcriptome sequencing and de novo assembly for a lichen species and describe the ongoing molecular processes and the most active pathways in C. rangiferina. This brings a meaningful contribution to publicly available lichen sequence information. These data provide a first glimpse into the molecular nature of the lichen symbiosis and characterise the transcriptional space of this remarkable organism. These data will also enable further studies aimed at deciphering the genetic mechanisms behind lichen desiccation tolerance.

Background The reindeer lichen is the product of a mutualistic relationship between a fungus and an algae. Lichen demonstrate a remarkable capacity to tolerate dehydration. This tolerance is driven by a variety of biochemical processes and the accumulation of specific secondary metabolites that may be of relevance to the pharmaceutical, biotechnology and agriculture industries. These protective metabolites hinder in vitro enzymatic reactions required in cDNA synthesis. Along with the low concentrations of RNA present within lichen tissues, the process of creating a cDNA library is technically challenging. Findings An evaluation of existing commercial and published protocols for RNA extraction from plant or fungal tissues has been performed and experimental conditions have been optimised to balance the need for the highest quality total ribonucleotides and the constraints of budget, time and human resources. Conclusion We present a protocol that balances inexpensive RNA extraction methods with commercial RNA clean-up kits to yield sufficient RNA for cDNA library construction. Evaluation of the protocol and the construction of, and sampling from, a cDNA library is used to demonstrate the suitability of the RNA extraction method for expressed sequence tag production.

Advances in plant genomics have permitted the analysis of several members of the grass family, including the major domesticated species, and provided new insights into the evolution of the major crops on earth. Two members, colonial bentgrass (Agrostis capillaris L.) and creeping bentgrass (A. stolonifera L.) have only recently been domesticated and provide an interesting case of polyploidy and comparison to crops that have undergone human selection for thousands of years. As an initial step of characterizing these genomes, we have sampled roughly 10% of their gene content, thereby also serving as a starting point for the construction of their physical and genetic maps. Sampling mRNA from plants subjected to environmental stress showed a remarkable increase in transcription of transposable elements. Both colonial and creeping bentgrass are allotetraploids and are considered to have one genome in common, designated the A2 genome. Analysis of conserved genes present among the ESTs suggests the colonial and creeping bentgrass A2 genomes diverged from a common ancestor approximately 2.2 million years ago (MYA), thereby providing an enhanced evolutionary zoom in respect to the origin of maize, which formed 4.8 MYA, and tetraploid wheat, which formed only 0.5 MYA and is the progenitor of domesticated hexaploid wheat.

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289-296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.

Background Butterfly wing color patterns are a key model for integrating evolutionary developmental biology and the study of adaptive morphological evolution. Yet, despite the biological, economical and educational value of butterflies they are still relatively under-represented in terms of available genomic resources. Here, we describe an Expression Sequence Tag (EST) project for Bicyclus anynana that has identified the largest available collection to date of expressed genes for any butterfly. Results By targeting cDNAs from developing wings at the stages when pattern is specified, we biased gene discovery towards genes potentially involved in pattern formation. Assembly of 9,903 ESTs from a subtracted library allowed us to identify 4,251 genes of which 2,461 were annotated based on BLAST analyses against relevant gene collections. Gene prediction software identified 2,202 peptides, of which 215 longer than 100 amino acids had no homology to any known proteins and, thus, potentially represent novel or highly diverged butterfly genes. We combined gene and Single Nucleotide Polymorphism (SNP) identification by constructing cDNA libraries from pools of outbred individuals, and by sequencing clones from the 3' end to maximize alignment depth. Alignments of multi-member contigs allowed us to identify over 14,000 putative SNPs, with 316 genes having at least one high confidence double-hit SNP. We furthermore identified 320 microsatellites in transcribed genes that can potentially be used as genetic markers. Conclusion Our project was designed to combine gene and sequence polymorphism discovery and has generated the largest gene collection available for any butterfly and many potential markers in expressed genes. These resources will be invaluable for exploring the potential of B. anynana in particular, and butterflies in general, as models in ecological, evolutionary, and developmental genetics.

Background Ginkgo biloba L. is the only surviving member of one of the oldest living seed plant groups with medicinal, spiritual and horticultural importance worldwide. As an evolutionary relic, it displays many characters found in the early, extinct seed plants and extant cycads. To establish a molecular base to understand the evolution of seeds and pollen, we created a cDNA library and EST dataset from the reproductive structures of male (microsporangiate), female (megasporangiate), and vegetative organs (leaves) of Ginkgo biloba. Results RNA from newly emerged male and female reproductive organs and immature leaves was used to create three distinct cDNA libraries from which 6,434 ESTs were generated. These 6,434 ESTs from Ginkgo biloba were clustered into 3,830 unigenes. A comparison of our Ginkgo unigene set against the fully annotated genomes of rice and Arabidopsis, and all available ESTs in Genbank revealed that 256 Ginkgo unigenes match only genes among the gymnosperms and non-seed plants – many with multiple matches to genes in non-angiosperm plants. Conversely, another group of unigenes in Gingko had highly significant homology to transcription factors in angiosperms involved in development, including MADS box genes as well as post-transcriptional regulators. Several of the conserved developmental genes found in Ginkgo had top BLAST homology to cycad genes. We also note here the presence of ESTs in G. biloba similar to genes that to date have only been found in gymnosperms and an additional 22 Ginkgo genes common only to genes from cycads. Conclusion Our analysis of an EST dataset from G. biloba revealed genes potentially unique to gymnosperms. Many of these genes showed homology to fully sequenced clones from our cycad EST dataset found in common only with gymnosperms. Other Ginkgo ESTs are similar to developmental regulators in higher plants. This work sets the stage for future studies on Ginkgo to better understand seed and pollen evolution, and to resolve the ambiguous phylogenetic relationship of G. biloba among the gymnosperms.

A Populus euphratica DNA microarray was constructed and used to analyze gene expression in trees growing in the desert. P. euphratica is shown to express a set of genes that is different from other Populus trees and these genes contribute to adaptation to saline growth conditions., Background Plants growing in their natural habitat represent a valuable resource for elucidating mechanisms of acclimation to environmental constraints. Populus euphratica is a salt-tolerant tree species growing in saline semi-arid areas. To identify genes involved in abiotic stress responses under natural conditions we constructed several normalized and subtracted cDNA libraries from control, stress-exposed and desert-grown P. euphratica trees. In addition, we identified several metabolites in desert-grown P. euphratica trees. Results About 14,000 expressed sequence tag (EST) sequences were obtained with a good representation of genes putatively involved in resistance and tolerance to salt and other abiotic stresses. A P. euphratica DNA microarray with a uni-gene set of ESTs representing approximately 6,340 different genes was constructed. The microarray was used to study gene expression in adult P. euphratica trees growing in the desert canyon of Ein Avdat in Israel. In parallel, 22 selected metabolites were profiled in the same trees. Conclusion Of the obtained ESTs, 98% were found in the sequenced P. trichocarpa genome and 74% in other Populus EST collections. This implies that the P. euphratica genome does not contain different genes per se, but that regulation of gene expression might be different and that P. euphratica expresses a different set of genes that contribute to adaptation to saline growth conditions. Also, all of the five measured amino acids show increased levels in trees growing in the more saline soil.

Development of composite inflorescences in the plant family Asteraceae has features that cannot be studied in the traditional model plants for flower development. InGerbera hybrida, inflorescences are composed of morphologically different types of flowers tightly packed into a flower head (capitulum). Individual floral organs such as pappus bristles (sepals) are developmentally specialized, stamens are aborted in marginal flowers, petals and anthers are fused structures, and ovaries are located inferior to other floral organs. These specific features have made gerbera a rewarding target of comparative studies. Here we report the analysis of a gerbera EST database containing 16,994 cDNA sequences. Comparison of the sequences with all plant peptide sequences revealed 1656 unique sequences for gerbera not identified elsewhere within the plant kingdom. Based on the EST database, we constructed a cDNA microarray containing 9000 probes and have utilized it in identification of flower-specific genes and abundantly expressed marker genes for flower scape, pappus, stamen, and petal development. Our analysis revealed several regulatory genes with putative functions in flower-organ development. We were also able to associate a number of abundantly and specifically expressed genes with flower-organ differentiation. Gerbera is an outcrossing species, for which genetic approaches to gene discovery are not readily amenable. However, reverse genetics with the help of gene transfer has been very informative. We demonstrate here the usability of the gerbera microarray as a reliable new tool for identifying novel genes related to specific biological questions and for large-scale gene expression analysis.

Motivation: Discovery of host and pathogen genes expressed at the plant--pathogen interface often requires the construction of mixed libraries that contain sequences from both genomes. Sequence identification requires high-throughput and reliable classification of genome origin. When using single-pass cDNA sequences difficulties arise from the short sequence length, the lack of sufficient taxonomically relevant sequence data in public databases and ambiguous sequence homology between plant and pathogen genes. Results: A novel method is described, which is independent of the availability of homologous genes and relies on subtle differences in codon usage between plant and fungal genes. We used support vector machines (SVMs) to identify the probable origin of sequences. SVMs were compared to several other machine learning techniques and to a probabilistic algorithm (PF-IND) for expressed sequence tag (EST) classification also based on codon bias differences. Our software (Eclat) has achieved a classification accuracy of 93.1% on a test set of 3217 EST sequences from Hordeum vulgare and Blumeria graminis, which is a significant improvement compared to PF-IND (prediction accuracy of 81.2% on the same test set). EST sequences with at least 50 nt of coding sequence can be classified using Eclat with high confidence. Eclat allows training of classifiers for any host--pathogen combination for which there are sufficient classified training sequences. Availability: Eclat is freely available on the Internet (http://mips.gsf.de/proj/est) or on request as a standalone version. Contact: friedel@informatik.uni-muenchen.de

To provide resources for barley genomics, 110,981 expressed sequence tags (ESTs) were generated from 22 cDNA libraries representing tissues at various developmental stages. This EST collection corresponds to approximately one-third of the 380,000 publicly available barley ESTs. Clustering and assembly resulted in 14,151 tentative consensi (TCs) and 11 073 singletons, altogether representing 25 224 putatively unique sequences. Of these, 17.5% showed no significant similarity to other barley ESTs present in dbEST. More than 41% of all barley genes are supposed to belong to multigene families and approximately 4% of the barley genes undergo alternative splicing. Based on the functional annotation of the set of unique sequences, the functional category 'Energy' was further analysed to reveal tissue- and stage-specific differences in gene expression. Hierarchical clustering of 362 differentially expressed TCs resulted in the identification of seven major clusters. The clusters reflect biochemical pathways predominantly activated in specific tissues and at various developmental stages. During seed germination glycolysis could be identified as the most predominant biochemical pathway. Germination-specific glycolysis is characterized by the coordinated expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase, whose antagonistic actions possibly regulate the flux of amino acids into protein biosynthesis and gluconeogenesis respectively. The expression of defence-related and antioxidant genes during germination might be controlled by the ethylene-signalling pathway as concluded from the coordinated expression of those genes and the transcription factors (TF) EIN3 and EREBPG. Moreover, because of their predominant expression in germinating seeds, TF of the AP2 and MYB type are presumably major regulators of germination.

Two million plant ESTs, from 20 different plant species, and totalling more than one 1000 Mbp of DNA sequence, represents a formidable transcriptomic resource. Sputnik uses the potential of this sequence resource to fill some of the information gap in the un-sequenced plant genomes and to serve as the foundation for in silicio comparative plant genomics. The complexity of the individual EST collections has been reduced using optimised EST clustering techniques. Annotation of cluster sequences is performed by exploiting and transferring information from the comprehensive knowledgebase already produced for the completed model plant genome (Arabidopsis thaliana) and by performing additional state of-the-art sequence analyses relevant to today's plant biologist. Functional predictions, comparative analyses and associative annotations for 500 000 plant EST derived peptides make Sputnik (http://mips.gsf.de/proj/sputnik/) a valid platform for contemporary plant genomics.

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.

During the last decade the small cruciferous plant Arabidopsis thaliana has become a model organism for flowering plants. Sequencing and analysis of the Arabidopsis genome is nearing completion. Beside an overview on methods and strategies for Arabidopsis genome analysis, a summary of the results from the first analysis is presented. This includes an overview on chromosomal organisation and topological features as well as a first comparison with other genomes.

Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Lichen is a symbiotic relationship between a fungus and an alga, and these organisms have a remarkable ability to survive in some of the harshest climates on earth. We report the de novo assembly and annotation of the transcriptome of the grey reindeer lichen, Cladonia rangiferina, using high-throughput next generation sequencing and traditional Sanger sequencing data. High quality sequence reads from a Roche GS FLX sequencing run and Sanger sequencing were de novo assembled. The genome of origin for the lichen sequences was predicted and the assembled sequences were annotated using BLASTX analysis against the non-redundant database. The C. rangiferina transcriptome was further characterised by functional annotation of the sequences against GO and KEGG databases. Our results present the first transcriptome sequencing and de novo assembly of any lichen species, describe the ongoing molecular processes and the most active pathways in C. rangiferina, and bring a significant increase to publicly available lichen sequence information.

Background: Plants growing in their natural habitat represent a valuable resource for elucidating mechanisms of acclimation to environmental constraints. Populus euphratica is a salt-tolerant tree species growing in saline semi-arid areas. To identify genes involved in abiotic stress responses under natural conditions we constructed several normalized and subtracted cDNA libraries from control, stress-exposed and desert-grown P. euphratica trees. In addition, we identified several metabolites in desert-grown P. euphratica trees. Results: About 14,000 expressed sequence tag (EST) sequences were obtained with a good representation of genes putatively involved in resistance and tolerance to salt and other abiotic stresses. A P. euphratica DNA microarray with a uni-gene set of ESTs representing approximately 6,340 different genes was constructed. The microarray was used to study gene expression in adult P. euphratica trees growing in the desert canyon of Ein Avdat in Israel. In parallel, 22 selected metabolites were profiled in the same trees. Conclusion: Of the obtained ESTs, 98% were found in the sequenced P. trichocarpa genome and 74% in other Populus EST collections. This implies that the P. euphratica genome does not contain different genes per se, but that regulation of gene expression might be different and that P. euphratica expresses a different set of genes that contribute to adaptation to saline growth conditions. Also, all of the five measured amino acids show increased levels in trees growing in the more saline soil.

Background Cycads are ancient seed plants (living fossils) with origins in the Paleozoic. Cycads are sometimes considered a 'missing link' as they exhibit characteristics intermediate between vascular non-seed plants and the more derived seed plants. Cycads have also been implicated as the source of 'Guam's dementia', possibly due to the production of S(+)-beta-methyl-alpha, beta-diaminopropionic acid (BMAA), which is an agonist of animal glutamate receptors.

Background Lichens are symbiotic organisms with a fungal and an algal or a cyanobacterial partner. Lichens inhabit some of the harshest climates on earth and most lichen species are desiccation-tolerant. Lichen desiccation-tolerance has been studied at the biochemical level and through proteomics, but the underlying molecular genetic mechanisms remain largely unexplored. The objective of our study was to examine the effects of dehydration and rehydration on the gene expression of Cladonia rangiferina. Results Samples of C. rangiferina were collected at several time points during both the dehydration and rehydration process and the gene expression intensities were measured using a custom DNA microarray. Several genes, which were differentially expressed in one or more time points, were identified. The microarray results were validated using qRT-PCR analysis. Enrichment analysis of differentially expressed transcripts was also performed to identify the Gene Ontology terms most associated with the rehydration and dehydration process. Conclusions Our data identify differential expression patterns for hundreds of genes that are modulated during dehydration and rehydration in Cladonia rangiferina. These dehydration and rehydration events clearly differ from each other at the molecular level and the largest changes to gene expression are observed within minutes following rehydration. Distinct changes are observed during the earliest stage of rehydration and the mechanisms not appear to be shared with the later stages of wetting or with drying. Several of the most differentially expressed genes are similar to genes identified in previous studies that have investigated the molecular mechanisms of other desiccation-tolerant organisms. We present here the first microarray experiment for any lichen species and have for the first time studied the genetic mechanisms behind lichen desiccation-tolerance at the whole transcriptome level.

Background 14-3-3 proteins have been implicated in many signalling mechanisms due to their interaction with Ser/Thr phosphorylated target proteins. They are evolutionarily well conserved in eukaryotic organisms from single celled protozoans and unicellular algae to plants and humans. A diverse array of target proteins has been found in higher plants and in human cell lines including proteins involved in cellular metabolism, apoptosis, cytoskeletal organisation, secretion and Ca2+ signalling. Results We found that the simple metazoan Hydra has four 14-3-3 isoforms. In order to investigate whether the diversity of 14-3-3 target proteins is also conserved over the whole animal kingdom we isolated 14-3-3 binding proteins from Hydra vulgaris using a 14-3-3-affinity column. We identified 23 proteins that covered most of the above-mentioned groups. We also isolated several novel 14-3-3 binding proteins and the Hydra specific secreted fascin-domain-containing protein PPOD. In addition, we demonstrated that one of the 14-3-3 isoforms, 14-3-3 HyA, interacts with one Hydra-Bcl-2 like protein in vitro. Conclusion Our results indicate that 14-3-3 proteins have been ubiquitous signalling components since the start of metazoan evolution. We also discuss the possibility that they are involved in the regulation of cell numbers in response to food supply in Hydra.

BackgroundAnthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development – when skeletogenesis is initiated, and symbionts are first acquired.ResultsOf 5081 unique peptide coding genes, 1084 were differentially expressed (P ≤ 0.05) in comparisons between four different stages of coral development, spanning key developmental transitions. Genes of likely relevance to the processes of settlement, metamorphosis, calcification and interaction with symbionts were characterised further and their spatial expression patterns investigated using whole-mount in situ hybridization.ConclusionThis study is the first large-scale investigation of developmental gene expression for any cnidarian, and has provided candidate genes for key roles in many aspects of coral biology, including calcification, metamorphosis and symbiont uptake. One surprising finding is that some of these genes have clear counterparts in higher animals but are not present in the closely-related sea anemoneNematostella. Secondly, coral-specific processes (i.e. traits which distinguish corals from their close relatives) may be analogous to similar processes in distantly related organisms. This first large-scale application of microarray analysis demonstrates the potential of this approach for investigating many aspects of coral biology, including the effects of stress and disease.