prot4EST: Translating Expressed Sequence Tags from neglected genomes

BACKGROUND: The genomes of an increasing number of species are being investigated through generation of expressed sequence tags (ESTs). However, ESTs are prone to sequencing errors and typically define incomplete transcripts, making downstream annotation difficult. Annotation would be greatly improved with robust polypeptide translations. Many current solutions for EST translation require a large number of full-length gene sequences for training purposes, a resource that is not available for the majority of EST projects. RESULTS: As part of our ongoing EST programs investigating these "neglected" genomes, we have developed a polypeptide prediction pipeline, prot4EST. It incorporates freely available software to produce final translations that are more accurate than those derived from any single method. We show that this integrated approach goes a long way to overcoming the deficit in training data. CONCLUSIONS: prot4EST provides a portable EST translation solution and can be usefully applied to >95% of EST projects to improve downstream annotation. It is freely available from

Ubiquitination screen using protein microarrays for comprehensive identification of Rsp5 substrates in yeast

Ubiquitin-protein ligases (E3s) are responsible for target recognition and regulate stability, localization or function of their substrates. However, the substrates of most E3 enzymes remain unknown. Here, we describe the development of a novel proteomic in vitro ubiquitination screen using a protein microarray platform that can be utilized for the discovery of substrates for E3 ligases on a global scale. Using the yeast E3 Rsp5 as a test system to identify its substrates on a yeast protein microarray that covers most of the yeast (Saccharomyces cerevisiae) proteome, we identified numerous known and novel ubiquitinated substrates of this E3 ligase. Our enzymatic approach was complemented by a parallel protein microarray protein interaction study. Examination of the substrates identified in the analysis combined with phage display screening allowed exploration of binding mechanisms and substrate specificity of Rsp5. The development of a platform for global discovery of E3 substrates is invaluable for understanding the cellular pathways in which they participate, and could be utilized for the identification of drug targets

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with ∼8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases

Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selection in parasitic nematodes and on how best to detect them. This study addresses these knowledge gaps by using field populations of Haemonchus contortus with well-defined benzimidazole treatment histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 genomic markers to genotype 20 individual adult worms from each of four H. contortus populations: two from closed sheep flocks with an approximate 20 year history of frequent benzimidazole treatment, and two populations with a history of little or no treatment. Sampling occurred in the same geographical region to limit genetic differentiation and maximise the detection sensitivity. A clear signature of selection was detected on chromosome I, centred on the isotype-1 β-tubulin gene. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I spanning 3.75 Mbp and 259 annotated genes, and one on chromosome II spanning a region of 3.3 Mbp and 206 annotated genes, including the isotype-2 β-tubulin locus. We also assessed how sensitivity was impacted by sequencing depth, worm number, and pooled versus individual worm sequence data. This study provides the first known direct genome-wide evidence for any parasitic nematode, that the isotype-1 β-tubulin gene is quantitatively the single most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole resistance loci of secondary importance. This study provides an experimental framework to maximise the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes

The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery

<p>Background: The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass drug administration to control parasitic nematodes. Its phylogenetic position makes it particularly well placed for comparison with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.</p> <p>Results: Here we report the detailed analysis of a draft genome assembly and extensive transcriptomic dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the most extensive transcriptomic dataset for any parasitic nematode reported to date. We show a general pattern of conservation of genome structure and gene content between H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes involved in parasite-specific pathways such as blood feeding, neurological function, and drug metabolism. In particular, we describe complete gene repertoires for known drug target families, providing the most comprehensive understanding yet of the action of several important anthelmintics. Also, we identify a set of genes enriched in the parasitic stages of the lifecycle and the parasite gut that provide a rich source of vaccine and drug target candidates.</p> <p>Conclusions: The H. contortus genome and transcriptome provides an essential platform for postgenomic research in this and other important strongylid parasites. </p&gt

NEMBASE4: the nematode transcriptome resource

AbstractNematode parasites are of major importance in human health and agriculture, and free-living species deliver essential ecosystem services. The genomics revolution has resulted in the production of many datasets of expressed sequence tags (ESTs) from a phylogenetically wide range of nematode species, but these are not easily compared. NEMBASE4 presents a single portal into extensively functionally annotated, EST-derived transcriptomes from over 60 species of nematodes, including plant and animal parasites and free-living taxa. Using the PartiGene suite of tools, we have assembled the publicly available ESTs for each species into a high-quality set of putative transcripts. These transcripts have been translated to produce a protein sequence resource and each is annotated with functional information derived from comparison with well-studied nematode species such as Caenorhabditis elegans and other non-nematode resources. By cross-comparing the sequences within NEMBASE4, we have also generated a protein family assignment for each translation. The data are presented in an openly accessible, interactive database. To demonstrate the utility of NEMBASE4, we have used the database to examine the uniqueness of the transcriptomes of major clades of parasitic nematodes, identifying lineage-restricted genes that may underpin particular parasitic phenotypes, possible viral pathogens of nematodes, and nematode-unique protein families that may be developed as drug targets

Radiation hybrid map spanning the huntington disease gene region of chromosome 4

Radiation hybrid (RH) mapping was used to construct a map of 11 markers in the distal 4 Mb of the short arm of chromosome 4, the region containing the Huntington disease gene. Two different methods for deriving the order of the markers were compared and both arrived at the same order as being the most likely. This order is also consistent with both the physical map constructed using pulsed-field gel electrophoresis (PFGE) and the meiotic linkage map. Comparing the RH map to the map determined by PFGE provided the means to equate RH map units (centirays) with actual physical distance in kilobases of DNA. In addition, a simple procedure for reducing the complexity of human DNA in radiation hybrids is described. One cell line isolated using this procedure contains, as its only human DNA, ~2 Mb surrounding the Huntington disease gene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29937/1/0000294.pd