Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

Addition of exogenous peptide sequences on viral capsids is a powerful approach to study the process of viral infection or to retarget viruses toward defined cell types. Until recently, it was not possible to manipulate the genome of mammalian reovirus and this was an obstacle to the addition of exogenous sequence tags onto the capsid of a replicating virus. This obstacle has now been overcome by the advent of the plasmid-based reverse genetics system. In the present study, reverse genetics was used to introduce different exogenous peptides, up to 40 amino acids long, at the carboxyl-terminal end of the σ1 outer capsid protein. The tagged viruses obtained were infectious, produce plaques of similar size, and could be easily propagated at hight titers. However, attempts to introduce a 750 nucleotides-long sequence failed, even when it was added after the stop codon, suggesting a possible size limitation at the nucleic acid level.L'addition aux capsides virales de séquences peptidiques exogènes est une approche puissante pour l'étude des processus d'infection virale ou pour cibler des virus vers des types cellulaires bien définis. Jusqu'à récemment, il n'était pas possible de manipuler le génome du réovirus de mammifères et ceci était un obstacle à l'addition de telles étiquettes exogènes sur la capside d'un virus pouvant se répliquer. Cet obstacle a maintenant été surmonté par la mise au point d'un système de génétique inverse à base de plasmides. Dans la présente étude, la génétique inverse a été utilisée pour introduire différents peptides exogènes, jusqu'à 40 acides aminés de longueur, à l'extrémité carboxyle de la protéine de capside externe σ1. Les virus obtenus étaient infectieux, produisent des plages de taille similaire, et peuvent être propagés facilement. Cependant, les efforts pour introduire une séquence de 750 nucléotides de longueur ont été infructueux, même lorsqu'un codon de terminaison était introduit avant celle-ci, suggérant une limite de taille au niveau des acides nucléiques.Conseil de recherches en sciences naturelles et en génie du Canad

A Dual Platform Approach to Transcript Discovery for the Planarian Schmidtea Mediterranea to Establish RNAseq for Stem Cell and Regeneration Biology

The use of planarians as a model system is expanding and the mechanisms that control planarian regeneration are being elucidated. The planarian Schmidtea mediterranea in particular has become a species of choice. Currently the planarian research community has access to this whole genome sequencing project and over 70,000 expressed sequence tags. However, the establishment of massively parallel sequencing technologies has provided the opportunity to define genetic content, and in particular transcriptomes, in unprecedented detail. Here we apply this approach to the planarian model system. We have sequenced, mapped and assembled 581,365 long and 507,719,814 short reads from RNA of intact and mixed stages of the first 7 days of planarian regeneration. We used an iterative mapping approach to identify and define de novo splice sites with short reads and increase confidence in our transcript predictions. We more than double the number of transcripts currently defined by publicly available ESTs, resulting in a collection of 25,053 transcripts described by combining platforms. We also demonstrate the utility of this collection for an RNAseq approach to identify potential transcripts that are enriched in neoblast stem cells and their progeny by comparing transcriptome wide expression levels between irradiated and intact planarians. Our experiments have defined an extensive planarian transcriptome that can be used as a template for RNAseq and can also help to annotate the S. mediterranea genome. We anticipate that suites of other 'omic approaches will also be facilitated by building on this comprehensive data set including RNAseq across many planarian regenerative stages, scenarios, tissues and phenotypes generated by RNAi

High through-put sequencing of the Parhyale hawaiensis mRNAs and microRNAs to aid comparative developmental studies

Understanding the genetic and evolutionary basis of animal morphological diversity will require comparative developmental studies that use new model organisms. This necessitates development of tools for the study of genetics and also the generation of sequence information of the organism to be studied. The development of next generation sequencing technology has enabled quick and cost effective generation of sequence information. Parhyale hawaiensis has emerged as a model organism of choice due to the development of advanced molecular tools, thus P. hawaiensis genetic information will help drive functional studies in this organism. Here we present a transcriptome and miRNA collection generated using next generation sequencing platforms. We generated approximately 1.7 million reads from a P. hawaiensis cDNA library constructed from embryos up to the germ band stage. These reads were assembled into a dataset comprising 163,501 transcripts. Using the combined annotation of Annot8r and pfam2go, Gene Ontology classifications was assigned to 20,597 transcripts. Annot8r was used to provide KEGG orthology to our transcript dataset. A total of 25,292 KEGG pathway assignments were defined and further confirmed with reciprocal blast against the NCBI nr protein database. This has identified many P. hawaiensis gene orthologs of key conserved signalling pathways involved in development. We also generated small RNA sequences from P. hawaiensis, identifying 55 conserved miRNAs. Sequenced small RNAs that were not annotated by stringent comparison to mirBase were used to search the Daphnia pulex for possible novel miRNAs. Using a conservative approach, we have identified 51 possible miRNA candidates conserved in the Daphnia pulex genome, which could be potential crustacean/arthropod specific miRNAs. Our study presents gene and miRNA discovery in a new model organism that does not have a sequenced genome. The data provided by our work will be valuable for the P. hawaiensis community as well as the wider evolutionary developmental biology community

Pou rouge de Californie et agrumiculture corse

National audienc

Pou rouge de Californie et agrumiculture corse

National audienc

454 read length distribution.

<p>A plot showing the distribution of the trimmed <i>P. hawaiensis</i> cDNA sequence reads generated from 454 Titanium sequencing chemistry.</p

The classifications of transcripts according to Gene Ontology (GO) terms.

<p>The classification counts are shown for each of the first tier terms of the three GO database domains; Cellular Component (A), Biological Process (B), and Molecular Function (C).</p

Conserved miRNAs with high expression values identified in <i>P. hawaiensis</i> developing embryos.

<p>The table lists 10 small RNA sequenced from <i>P. hawaiensis</i> embryos with identified homology to known miRNA sequences present in mirBase (release 17). Bases in bold and lower case indicate the allowed mismatch during homology mapping. Tag counts refer to the number of sequence matches found to that particular reference miRNA sequence. Only the sequences greater than 100 tag counts for either of the two stages examined has been listed. All identified miRNAs are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033784#pone.0033784.s004" target="_blank">Tables S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033784#pone.0033784.s005" target="_blank">S5</a>.</p

MapMi homology mapping of <i>P. hawaiensis</i> conserved miRNA to the <i>Daphnia pulex</i> genome.

<p>Table shows the result of MapMi homology mapping to the <i>D. pulex</i> genome with <i>P. hawaiensis</i> sequences annotated by known miRNA from mirBase with a tolerance of a 1 bp mismatch. The mapping position on the <i>D. pulex</i> chromosome corresponding to known miRNA sequences are shown with the calculated MapMi score for the mapping and RNA fold analysis.</p