Simon J. Hubbard

A Comprehensive Collection of Chicken cDNAs

Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank. We describe a large-scale expressed-sequence-tag (EST) project aimed at gene discovery in chickens (http://www.chick.umist.ac.uk). In total, 339,314 ESTs have been sequenced from 64 cDNA libraries generated from 21 different embryonic and adult tissues. These were clustered and assembled into 85,486 contiguous sequences (contigs). We find that a minimum of 38% of the contigs have orthologs in other organisms and define an upper limit of 13,000 new chicken genes. The remaining contigs may include novel avian specific or rapidly evolving genes. Comparison of the contigs with known chicken genes and orthologs indicates that 30% include cDNAs that contain the start codon and 20% of the contigs represent full-length cDNA sequences. Using this dataset, we estimate that chickens have approximately 35,000 genes in total, suggesting that this number may be a characteristic feature of vertebrates.

Global Translational Responses to Oxidative Stress Impact upon Multiple Levels of Protein Synthesis

Global inhibition of protein synthesis is a common response to stress conditions. We have analyzed the regulation of protein synthesis in response to oxidative stress induced by exposure to H2O2 in the yeast Saccharomyces cerevisiae. Our data show that H2O2 causes an inhibition of translation initiation dependent on the Gcn2 protein kinase, which phosphorylates the α-subunit of eukaryotic initiation factor-2. Additionally, our data indicate that translation is regulated in a Gcn2-independent manner because protein synthesis was still inhibited in response to H2O2 in a gcn2 mutant. Polysome analysis indicated that H2O2 causes a slower rate of ribosomal runoff, consistent with an inhibitory effect on translation elongation or termination. Furthermore, analysis of ribosomal transit times indicated that oxidative stress increases the average mRNA transit time, confirming a post-initiation inhibition of translation. Using microarray analysis of polysome- and monosome-associated mRNA pools, we demonstrate that certain mRNAs, including mRNAs encoding stress protective molecules, increase in association with ribosomes following H2O2 stress. For some candidate mRNAs, we show that a low concentration of H2O2 results in increased protein production. In contrast, a high concentration of H2O2 promotes polyribosome association but does not necessarily lead to increased protein production. We suggest that these mRNAs may represent an mRNA store that could become rapidly activated following relief of the stress condition. In summary, oxidative stress elicits complex translational reprogramming that is fundamental for adaptation to the stress.

A systematic approach to modeling, capturing, and disseminating proteomics experimental data

Both the generation and the analysis of proteome data are becoming increasingly widespread, and the field of proteomics is moving incrementally toward high-throughput approaches. Techniques are also increasing in complexity as the relevant technologies evolve. A standard representation of both the methods used and the data generated in proteomics experiments, analogous to that of the MIAME (minimum information about a microarray experiment) guidelines for transcriptomics, and the associated MAGE (microarray gene expression) object model and XML (extensible markup language) implementation, has yet to emerge. This hinders the handling, exchange, and dissemination of proteomics data. Here, we present a UML (unified modeling language) approach to proteomics experimental data, describe XML and SQL (structured query language) implementations of that model, and discuss capture, storage, and dissemination strategies. These make explicit what data might be most usefully captured about proteomics experiments and provide complementary routes toward the implementation of a proteome repository.

The chicken as a model for large-scale analysis of vertebrate gene function.

The chicken has been an important experimental system for developmental biology, immunology and microbiology, having led to many fundamental discoveries. The increase in genomic resources, easy access to the embryo and the application of RNA interference mean that it will be easy and quick to use chick embryos to screen the function of many genes during embryonic development. So, it seems likely that the chicken will increasingly be the system of choice for many vertebrate biologists who are interested in gene function.

Comparative genome analysis of filamentous fungi reveals gene family expansions associated with fungal pathogenesis.

Fungi and oomycetes are the causal agents of many of the most serious diseases of plants. Here we report a detailed comparative analysis of the genome sequences of thirty-six species of fungi and oomycetes, including seven plant pathogenic species, that aims to explore the common genetic features associated with plant disease-causing species. The predicted translational products of each genome have been clustered into groups of potential orthologues using Markov Chain Clustering and the data integrated into the e-Fungi object-oriented data warehouse (http://www.e-fungi.org.uk/). Analysis of the species distribution of members of these clusters has identified proteins that are specific to filamentous fungal species and a group of proteins found only in plant pathogens. By comparing the gene inventories of filamentous, ascomycetous phytopathogenic and free-living species of fungi, we have identified a set of gene families that appear to have expanded during the evolution of phytopathogens and may therefore serve important roles in plant disease. We have also characterised the predicted set of secreted proteins encoded by each genome and identified a set of protein families which are significantly over-represented in the secretomes of plant pathogenic fungi, including putative effector proteins that might perturb host cell biology during plant infection. The results demonstrate the potential of comparative genome analysis for exploring the evolution of eukaryotic microbial pathogenesis.

BioMart Central Portal: an open database network for the biological community

BioMart Central Portal is a first of its kind, community-driven effort to provide unified access to dozens of biological databases spanning genomics, proteomics, model organisms, cancer data, ontology information and more. Anybody can contribute an independently maintained resource to the Central Portal, allowing it to be exposed to and shared with the research community, and linking it with the other resources in the portal. Users can take advantage of the common interface to quickly utilize different sources without learning a new system for each. The system also simplifies cross-database searches that might otherwise require several complicated steps. Several integrated tools streamline common tasks, such as converting between ID formats and retrieving sequences. The combination of a wide variety of databases, an easy-to-use interface, robust programmatic access and the array of tools make Central Portal a one-stop shop for biological data querying. Here, we describe the structure of Central Portal and show example queries to demonstrate its capabilities. Database URL: http://central.biomart.org.

The mzIdentML Data Standard for Mass Spectrometry-Based Proteomics Results

We report the release of mzIdentML, an exchange standard for peptide and protein identification data, designed by the Proteomics Standards Initiative. The format was developed by the Proteomics Standards Initiative in collaboration with instrument and software vendors, and the developers of the major open-source projects in proteomics. Software implementations have been developed to enable conversion from most popular proprietary and open-source formats, and mzIdentML will soon be supported by the major public repositories. These developments enable proteomics scientists to start working with the standard for exchanging and publishing data sets in support of publications and they provide a stable platform for bioinformatics groups and commercial software vendors to work with a single file format for identification data.

Differential Expression of Ion Channel Transcripts in Atrial Muscle and Sinoatrial Node in Rabbit

The aim of the study was to identify ion channel transcripts expressed in the sinoatrial node (SAN), the pacemaker of the heart. Functionally, the SAN can be divided into central and peripheral regions (center is adapted for pacemaking only, whereas periphery is adapted to protect center and drive atrial muscle as well as pacemaking) and the aim was to study expression in both regions. In rabbit tissue, the abundance of 30 transcripts (including transcripts for connexin, Na+, Ca2+, hyperpolarization-activated cation and K+ channels, and related Ca2+ handling proteins) was measured using quantitative PCR and the distribution of selected transcripts was visualized using in situ hybridization. Quantification of individual transcripts (quantitative PCR) showed that there are significant differences in the abundance of 63% of the transcripts studied between the SAN and atrial muscle, and cluster analysis showed that the transcript profile of the SAN is significantly different from that of atrial muscle. There are apparent isoform switches on moving from atrial muscle to the SAN center: RYR2 to RYR3, Nav1.5 to Nav1.1, Cav1.2 to Cav1.3 and Kv1.4 to Kv4.2. The transcript profile of the SAN periphery is intermediate between that of the SAN center and atrial muscle. For example, Nav1.5 messenger RNA is expressed in the SAN periphery (as it is in atrial muscle), but not in the SAN center, and this is probably related to the need of the SAN periphery to drive the surrounding atrial muscle.

The Functional Genomics Experiment model (FuGE): an extensible framework for standards in functional genomics

The Functional Genomics Experiment data model (FuGE) has been developed to facilitate convergence of data standards for high-throughput, comprehensive analyses in biology. FuGE models the components of an experimental activity that are common across different technologies, including protocols, samples and data. FuGE provides a foundation for describing entire laboratory workflows and for the development of new data formats. The Microarray Gene Expression Data society and the Proteomics Standards Initiative have committed to using FuGE as the basis for defining their respective standards, and other standards groups, including the Metabolomics Standards Initiative, are evaluating FuGE in their development efforts. Adoption of FuGE by multiple standards bodies will enable uniform reporting of common parts of functional genomics workflows, simplify data-integration efforts and ease the burden on researchers seeking to fulfill multiple minimum reporting requirements. Such advances are important for transparent data management and mining in functional genomics and systems biology.

Global gene expression profiling reveals widespread yet distinctive translational responses to different eukaryotic translation initiation factor 2B-targeting stress pathways

ABSTRACT Global inhibition of protein synthesis is a hallmark of many cellular stress conditions. Even though specific mRNAs defy this (e.g., yeast GCN4 and mammalian ATF4), the extent and variation of such resistance remain uncertain. In this study, we have identified yeast mRNAs that are translationally maintained following either amino acid depletion or fusel alcohol addition. Both stresses inhibit eukaryotic translation initiation factor 2B, but via different mechanisms. Using microarray analysis of polysome and monosome mRNA pools, we demonstrate that these stress conditions elicit widespread yet distinct translational reprogramming, identifying a fundamental role for translational control in the adaptation to environmental stress. These studies also highlight the complex interplay that exists between different stages in the gene expression pathway to allow specific preordained programs of proteome remodeling. For example, many ribosome biogenesis genes are coregulated at the transcriptional and translational levels following amino acid starvation. The transcriptional regulation of these genes has recently been connected to the regulation of cellular proliferation, and on the basis of our results, the translational control of these mRNAs should be factored into this equation.