Requirements for translation re-initiation in Escherichia coli: roles of initiator tRNA and initiation factors IF2 and IF3

Despite its importance in post-transcriptional regulation of polycistronic operons in Escherichia coli, little is known about the mechanism of translation re-initiation, which occurs when the same ribosome used to translate an upstream open reading frame (ORF) also translates a downstream ORF. To investigate translation re-initiation in Escherichia coli, we constructed a di-cistronic reporter in which a firefly luciferase gene was linked to a chloramphenicol acetyltransferase gene using a segment of the translationally coupled geneV–geneVII intercistronic region from M13 phage. With this reporter and mutant initiator tRNAs, we show that two of the unique properties of E. coli initiator tRNA – formylation of the amino acid attached to the tRNA and binding of the tRNA to the ribosomal P-site – are as important for re-initiation as for de novo initiation. Overexpression of IF2 or increasing the affinity of mutant initiator tRNA for IF2 enhanced re-initiation efficiency, suggesting that IF2 is required for efficient re-initiation. In contrast, overexpression of IF3 led to a marked decrease in re-initiation efficiency, suggesting that a 30S ribosome and not a 70S ribosome is used for translation re-initiation. Strikingly, overexpression of IF3 also blocked E. coli from acting as a host for propagation of M13 phage

Integrating Text Mining into the MGI Biocuration Workflow

A major challenge for the development of resources for functional and comparative genomics is the extraction of data from the biomedical literature. Although text retrieval and extraction for biological data is an active research field, few applications have been integrated into production literature curation systems such as those of the model organism databases.

In September 2008, Mouse Genome Informatics (MGI) at The Jackson Lab initiated a search for dictionary-based text mining tools that we could integrate into our curation workflow. MGI has rigorous document triage and annotation procedures designed to identify articles about mouse genome biology and determine whether those articles should be curated. We currently screens approximately 1000 journal articles a month for Gene Ontology terms, gene mapping, gene expression, phenotype data and other key biological information. Although we don’t foresee that human curation tasks can be fully automated in the near future, we are eager to implement entity name recognition and gene tagging tools that can help streamline our curation workflow and simplify gene indexing tasks in the MGI system. 

In this presentation, we discuss our search process and the steps we took to identify a short list of potential tools for further evaluation. We present our performance metrics and success criteria, and pilot projects in progress. The primary applications under current review are Fraunhofer SCAI’s ProMiner and NCBO’s Open-Biomedical Annotator. 
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The effect of consecutive days of exercise on markers of oxidative stress

We examined the influence of 3 consecutive days of high-intensity cycling on blood and urinary markers of oxidative stress. Eight highly-trained male cyclists (VO2 max 76 +/- 4 mL.kg-1.min-1; mean +/- SD) completed an interval session (9 exercise bouts lasting 30 s each, at 150% peak power output) on day 1, followed by 2 laboratory-simulated 30 km time trials on days 2 and 3. The cyclists also completed a submaximal exercise trial matched to the interval session for oxygen consumption. Blood was collected pre- and post-exercise for the determination of malondialdehyde (MDA), total antioxidant status (TAS), vitamin E, and the antioxidant enzyme activity of superoxide dismutase and glutathione peroxidase, while urine was collected for the determination of allantoin. There were significant increases in plasma MDA concentrations (p < 0.01), plasma TAS (p < 0.01), and urinary allantoin excretion (p < 0.01) following the high-intensity interval session on day 1, whereas plasma vitamin E concentration significantly decreased (p = 0.028). Post-exercise changes in plasma MDA (p = 0.036), TAS concentrations (p = 0.039), and urinary allantoin excretion (p = 0.031) were all significantly attenuated over the 3 consecutive days of exercise, whereas resting plasma TAS concentration was elevated. There were no significant changes in plasma MDA, TAS, or allantoin excretion following submaximal exercise and there were no significant changes in antioxidant enzyme activity over consecutive days of exercise or following submaximal exercise. Consecutive days of high-intensity exercise enhanced resting plasma TAS concentration and reduced the post-exercise increase in plasma MDA concentrations