Stephen Rudd

MIPS: a database for genomes and protein sequences

The Munich Information Center for Protein Sequences (MIPS-GSF, Neuherberg, Germany) continues to provide genome-related information in a systematic way. MIPS supports both national and European sequencing and functional analysis projects, develops and maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences, and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the databases for the comprehensive set of genomes (PEDANT genomes), the database of annotated human EST clusters (HIB), the database of complete cDNAs from the DHGP (German Human Genome Project), as well as the project specific databases for the GABI (Genome Analysis in Plants) and HNB (Helmholtz-Netzwerk Bioinformatik) networks. The Arabidospsis thaliana database (MATDB), the database of mitochondrial proteins (MITOP) and our contribution to the PIR International Protein Sequence Database have been described elsewhere [Schoof et al. (2002) Nucleic Acids Res., 30, 91-93; Scharfe et al. (2000) Nucleic Acids Res., 28, 155-158; Barker et al. (2001) Nucleic Acids Res., 29, 29-32]. All databases described, the protein analysis tools provided and the detailed descriptions of our projects can be accessed through the MIPS World Wide Web server (http://mips.gsf.de).

Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert

BackgroundPlants 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.ResultsAbout 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.ConclusionOf 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.

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.

Microarray analysis identifies candidate genes for key roles in coral development

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 anemone Nematostella. 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.

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR.

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function.

Sputnik: a database platform for comparative plant genomics

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 todays 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.

Genome-Wide in Silico Mapping of Scaffold/Matrix Attachment Regions in Arabidopsis Suggests Correlation of Intragenic Scaffold/Matrix Attachment Regions with Gene Expression

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.

PlantMarkers—a database of predicted molecular markers from plants

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.

Spatiotemporal Expression Control Correlates with Intragenic Scaffold Matrix Attachment Regions (S/MARs) in Arabidopsis thaliana

Scaffold/matrix attachment regions (S/MARs) are essential for structural organization of the chromatin within the nucleus and serve as anchors of chromatin loop domains. A significant fraction of genes in Arabidopsis thaliana contains intragenic S/MAR elements and a significant correlation of S/MAR presence and overall expression strength has been demonstrated. In this study, we undertook a genome scale analysis of expression level and spatiotemporal expression differences in correlation with the presence or absence of genic S/MAR elements. We demonstrate that genes containing intragenic S/MARs are prone to pronounced spatiotemporal expression regulation. This characteristic is found to be even more pronounced for transcription factor genes. Our observations illustrate the importance of S/MARs in transcriptional regulation and the role of chromatin structural characteristics for gene regulation. Our findings open new perspectives for the understanding of tissue- and organ-specific regulation of gene expression.

EST analysis in Ginkgo biloba: An assessment of conserved developmental regulators and gymnosperm specific genes

BackgroundGinkgo 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.ResultsRNA 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.ConclusionOur 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.