Karl-Heinz Glatting

Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress

BackgroundBrown algae (Phaeophyceae) are phylogenetically distant from red and green algae and an important component of the coastal ecosystem. They have developed unique mechanisms that allow them to inhabit the intertidal zone, an environment with high levels of abiotic stress. Ectocarpus siliculosus is being established as a genetic and genomic model for the brown algal lineage, but little is known about its response to abiotic stress.ResultsHere we examine the transcriptomic changes that occur during the short-term acclimation of E. siliculosus to three different abiotic stress conditions (hyposaline, hypersaline and oxidative stress). Our results show that almost 70% of the expressed genes are regulated in response to at least one of these stressors. Although there are several common elements with terrestrial plants, such as repression of growth-related genes, switching from primary production to protein and nutrient recycling processes, and induction of genes involved in vesicular trafficking, many of the stress-regulated genes are either not known to respond to stress in other organisms or are have been found exclusively in E. siliculosus.ConclusionsThis first large-scale transcriptomic study of a brown alga demonstrates that, unlike terrestrial plants, E. siliculosus undergoes extensive reprogramming of its transcriptome during the acclimation to mild abiotic stress. We identify several new genes and pathways with a putative function in the stress response and thus pave the way for more detailed investigations of the mechanisms underlying the stress tolerance ofbrown algae.

W2H: WWW interface to the GCG sequence analysis package.

MOTIVATION The user-friendly, graphical X-windows interface (WPI) to the GCG sequence analysis package can often not be used due to the lack of an X-server on PC or Macintosh computers. Because Web browsers like Netscape are much more common on those platforms, we decided to develop W2H, a WWW interface to the GCG Sequence Analysis Software Package with nearly the same functionality as the X-windows interface WPI. RESULTS The new WWW interface (W2H) to the GCG Sequence Analysis Software Package (Wisconsin Package) supports modern Web technologies, like client-pull method, or embedded scripting language, and provides a reasonable platform independence. The interface is quite comprehensive with advanced features like sequence selector, search set builder, enzyme chooser, access to sequence databases, uploading client files to the GCG server or displaying and manipulating graphical outputs in addition to GCG analysis programs. W2H also manages secure access to both GCG server and user data. For special environments, like workshops, conferences and company intranets, there is a special mode (Intranet mode) with less security constraints. The behaviour of W2H is mostly controlled by meta-data files describing the applications and giving a base for dynamic creation of HTML documents. This paper presents mainly the development approaches used, and architectural design aspects of W2H. AVAILABILITY W2H is available by ftp://ftp.ebi.ac. uk/pub/software/unix/w2h or ftp://genome.dkfz-heidelberg.de/pub/w2h CONTACT m.senger@ebi.ac.uk

ESTAnnotator: a tool for high throughput EST annotation

In high throughput sequence analysis, it is often necessary to combine the results of contemporary bioinformatics tools, because no individual tool alone computes all the requested information. ESTAnnotator is a tool for the high throughput annotation of expressed sequence tags (ESTs) by automatically running a collection of bioinformatics applications. In the first step, a quality check is performed and repeats, vector parts and low quality sequences are masked. Then successive steps of database searching and EST clustering are performed. Already known transcripts present within mRNA and genomic DNA reference databases are identified. Subsequently, tools for the clustering of anonymous ESTs, and for further database searches at the protein level, are applied. Finally, the outputs of each individual tool are gathered and the relevant results presented in a descriptive summary. ESTAnnotator was already successfully applied for the systematic identification and characterisation of novel human genes involved in cartilage/bone formation, growth, differentiation and homeostasis. ESTAnnotator is available at http://genome.dkfz-heidelberg.de, contact: genome@dkfz.de.

New miRNAs cloned from neuroblastoma

BackgroundMicroRNAs (miRNAs) are a novel class of gene expression regulators implicated in cancer biology. Neuroblastoma (NB) is an embryonal tumour consisting of neural crest-derived undifferentiated cells and is characterised by variable clinical courses ranging from spontaneous regression to therapy-resistant progression. Recent advances identified a subset of miRNAs with putative function in NB biology. However, the full repertoire of miRNAs expressed in NBs is not available.ResultsWe describe miRNA profiles of 13 NB specimens and 2 NB cell lines as determined by miRNA cloning. A total of 3153 sequences were sequenced and analysed by a miRNA prediction tool (miRpredict). Our library covered 27% miRNAs known to date. 39 reads corresponding to 25 individual sequences were classified as novel miRNAs, including miRNA* species of 10 known miRNAs. Expression of 5 new miRNA* forms and 8 individual sequences was supported by Northern blotting. Most of the novel miRNA genes are not related to each other and do not share homology with the annotated sequences in the public miRNA database, but they are conserved within mammals or have close homologues in primates genomes.ConclusionWe provide evidence for 29 new miRNA and miRNA-like sequences (24 novel sequences and 5 miRNAs discovered initially in other species). Some of these newly identified sequences reside within frequently altered chromosomal regions in NB tumours and may play a role in NB biology.

A task framework for the web interface W2H

SUMMARY The W3H task framework allows the execution of compound jobs utilizing the description of work and data flows in a heterogeneous bioinformatics environment using meta-data information. By means of these descriptions, the task system can schedule the necessary execution of applications available in the environment, depending on rules specified in the meta-data. By integrating this task framework into the publicly available web interface W2H, similarly based on meta-data, web access and data management are immediately available for each task description. Authors of task descriptions can base their work on the underlying classes and objects to be able to describe dependency rules between previously independent applications. The result of a compound task is given as XML data that is translated according to XSLT data into web pages or plain text to report the result of the task to the user. AVAILABILITY Within the HUSAR environment at DKFZ http://genome.dkfz-heidelberg.de/

Genome-wide prediction of splice-modifying SNPs in human genes using a new analysis pipeline called AASsites

BackgroundSome single nucleotide polymorphisms (SNPs) are known to modify the risk of developing certain diseases or the reaction to drugs. Due to next generation sequencing methods the number of known human SNPs has grown. Not all SNPs lead to a modified protein, which may be the origin of a disease. Therefore, the recognition of functional SNPs is needed. Because most SNP annotation tools look for SNPs which lead to an amino acid exchange or a premature stop, we designed a new tool called AASsites which searches for SNPs which modify splicing.ResultsAASsites uses several gene prediction programs and open reading frame prediction to compare the wild type (wt) and the variant gene sequence. The results of the comparison are combined by a handmade rule system to classify a change in splicing as “likely, probable, unlikely”. Having received good results from tests with SNPs known for changing the splicing pattern we checked 80,000 SNPs from the human genome which are located near splice sites for their ability to change the splicing pattern of the gene and hereby result in a different protein. We identified 301 “likely” and 985 “probable” classified SNPs with such characteristics. Within this set 33 SNPs are described in the ssSNP Target database to cause modified splicing.ConclusionsWith AASsites single SNPs can be checked for those causing splice modifications. Screening 80,000 known human SNPs we detected about 1,200 SNPs which probably modify splicing. AASsites is available at http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar using any web browser.

ProtSweep, 2Dsweep and DomainSweep: protein analysis suite at DKFZ

The wealth of transcript information that has been made publicly available in recent years has led to large pools of individual web sites offering access to bioinformatics software. However, finding out which services exist, what they can or cannot do, how to use them and how to feed results from one service to the next one in the right format can be very time and resource consuming, especially for non-experts. Automating this task, we present a suite of protein annotation pipelines (tasks) developed at the German Cancer Research Centre (DKFZ) oriented to protein annotation by homology (ProtSweep), by domain analysis (DomainSweep), and by secondary structure elements (2Dsweep). The aim of these tasks is to perform an exhaustive structural and functional analysis employing a wide variety of methods in combination with the most updated public databases. The three servers are available for academic users at the HUSAR open server http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar/

cDNA2Genome: A tool for mapping and annotating cDNAs

BackgroundIn the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problematic cDNAs in order to speed up the posterior annotation process.ResultscDNA2Genome is an application for the automatic high-throughput mapping and characterization of cDNAs. It utilizes current annotation data and the most up to date databases, especially in the case of ESTs and mRNAs in conjunction with a vast number of approaches to gene prediction in order to perform a comprehensive assessment of the cDNA exon-intron structure. The final result of cDNA2Genome is an XML file containing all relevant information obtained in the process. This XML output can easily be used for further analysis such us program pipelines, or the integration of results into databases. The web interface to cDNA2Genome also presents this data in HTML, where the annotation is additionally shown in a graphical form. cDNA2Genome has been implemented under the W3H task framework which allows the combination of bioinformatics tools in tailor-made analysis task flows as well as the sequential or parallel computation of many sequences for large-scale analysis.ConclusionscDNA2Genome represents a new versatile and easily extensible approach to the automated mapping and annotation of human cDNAs. The underlying approach allows sequential or parallel computation of sequences for high-throughput analysis of cDNAs.

Genome-Wide Analysis of the Circulating miRNome After Cerebral Ischemia Reveals a Reperfusion-Induced MicroRNA Cluster

Background and Purpose— Circulating microRNAs (miRNAs) are emerging biomarkers for stroke because of their high stability in the bloodstream and association with pathophysiologic conditions. However, the circulating whole-genome miRNAs (miRNome) has not been characterized comprehensively in the acute phase of stroke. Methods— We profiled the circulating miRNome in mouse models of acute ischemic and hemorrhagic stroke by next-generation sequencing. Stroke models were compared with sham-operated and naive mice to identify deregulated circulating miRNAs. Top-ranked miRNAs were validated and further characterized by quantitative reverse transcription polymerase chain reaction. Results— We discovered 24 circulating miRNAs with an altered abundance in the circulation 3 hours after ischemia, whereas the circulating miRNome was not altered after intracerebral hemorrhage compared with sham-operated mice. Among the upregulated miRNAs in ischemia, the top-listed miR-1264/1298/448 cluster was strongly dependent on reperfusion in different ischemia models. A time course experiment revealed that the miR-1264/1298/448 cluster peaked in the circulation around 3 hours after reperfusion and gradually decreased thereafter. Conclusions— Alteration of the miRNome in the circulation is associated with cerebral ischemia/reperfusion, but not hemorrhage, suggesting a potential to serve as biomarkers for reperfusion in the acute phase. The pathophysiological role of reperfusion-inducible miR-1264/1298/448 cluster, which is located on chromosome X within the introns of the serotonin receptor HTR2C, requires further investigation.

PATH: a task for the inference of phylogenies

UNLABELLED Phylogenetic Analysis Task in Husar (PATH) is a task for the inference of phylogenies. It executes three phylogenetic methods and automatically chooses the evolutionary model for each set of data. The output of the tasks shows the consensus trees together with full results obtained from all executed methods. AVAILABILITY PATH is available at the German EMBnet node after registration via www at http://genome.dkfz-heidelberg.de