urg1: a uracil-regulatable promoter system for fission yeast with short induction and repression times.

BACKGROUND: The fission yeast Schizosaccharomyces pombe is a popular genetic model organism with powerful experimental tools. The thiamine-regulatable nmt1 promoter and derivatives, which take >15 hours for full induction, are most commonly used for controlled expression of ectopic genes. Given the short cell cycle of fission yeast, however, a promoter system that can be rapidly regulated, similar to the GAL system for budding yeast, would provide a key advantage for many experiments. METHODOLOGY/PRINCIPAL FINDINGS: We used S. pombe microarrays to identify three neighbouring genes (urg1, urg2, and urg3) whose transcript levels rapidly and strongly increased in response to uracil, a condition which otherwise had little effect on global gene expression. We cloned the promoter of urg1 (uracil-regulatable gene) to create several PCR-based gene targeting modules for replacing native promoters with the urg1 promoter (Purg1) in the normal chromosomal locations of genes of interest. The kanMX6 and natMX6 markers allow selection under urg1 induced and repressed conditions, respectively. Some modules also allow N-terminal tagging of gene products placed under urg1 control. Using pom1 as a proof-of-principle, we observed a maximal increase of Purg1-pom1 transcripts after uracil addition within less than 30 minutes, and a similarly rapid decrease after uracil removal. The induced and repressed transcriptional states remained stable over 24-hour periods. RT-PCR comparisons showed that both induced and repressed Purg1-pom1 transcript levels were lower than corresponding P3nmt1-pom1 levels (wild-type nmt1 promoter) but higher than P81nmt1-pom1 levels (weak nmt1 derivative). CONCLUSIONS/SIGNIFICANCE: We exploited the urg1 promoter system to rapidly induce pom1 expression at defined cell-cycle stages, showing that ectopic pom1 expression leads to cell branching in G2-phase but much less so in G1-phase. The high temporal resolution provided by the urg1 promoter should facilitate experimental design and improve the genetic toolbox for the fission yeast community

Spectral analysis of Gene co-expression network of Zebrafish

We analyze the gene expression data of Zebrafish under the combined framework of complex networks and random matrix theory. The nearest neighbor spacing distribution of the corresponding matrix spectra follows random matrix predictions of Gaussian orthogonal statistics. Based on the eigenvector analysis we can divide the spectra into two parts, first part for which the eigenvector localization properties match with the random matrix theory predictions, and the second part for which they show deviation from the theory and hence are useful to understand the system dependent properties. Spectra with the localized eigenvectors can be characterized into three groups based on the eigenvalues. We explore the position of localized nodes from these different categories. Using an overlap measure, we find that the top contributing nodes in the different groups carry distinguished structural features. Furthermore, the top contributing nodes of the different localized eigenvectors corresponding to the lower eigenvalue regime form different densely connected structure well separated from each other. Preliminary biological interpretation of the genes, associated with the top contributing nodes in the localized eigenvectors, suggests that the genes corresponding to same vector share common features.Comment: 6 pages, four figures (accepted in EPL

Site Layout of the proposed new Hadrons' Injector Chain at CERN

The replacement of almost all the LHC injector complex on the Meyrin-site of CERN (Linac2, PSB and PS) is planned within the next 10 years. The layout foreseen for the new accelerators is described in this paper, together with its compatibility with the existing experimental physics facilities. These machines can, after upgrade, supply with high beam power future physics facilities for radioactive ions and/or neutrinos. Their possible layout is also sketched in this document

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein “DDOR” (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications

Kinetic modelling of competition and depletion of shared miRNAs by competing endogenous RNAs

Non-conding RNAs play a key role in the post-transcriptional regulation of mRNA translation and turnover in eukaryotes. miRNAs, in particular, interact with their target RNAs through protein-mediated, sequence-specific binding, giving rise to extended and highly heterogeneous miRNA-RNA interaction networks. Within such networks, competition to bind miRNAs can generate an effective positive coupling between their targets. Competing endogenous RNAs (ceRNAs) can in turn regulate each other through miRNA-mediated crosstalk. Albeit potentially weak, ceRNA interactions can occur both dynamically, affecting e.g. the regulatory clock, and at stationarity, in which case ceRNA networks as a whole can be implicated in the composition of the cell's proteome. Many features of ceRNA interactions, including the conditions under which they become significant, can be unraveled by mathematical and in silico models. We review the understanding of the ceRNA effect obtained within such frameworks, focusing on the methods employed to quantify it, its role in the processing of gene expression noise, and how network topology can determine its reach.Comment: review article, 29 pages, 7 figure

Linac4 Technical Design Report

Linac4 is an H- linear accelerator, intended to replace Linac2 as injector to the PS Booster (PSB). By delivering to the PSB a beam at 160 MeV energy, Linac4 will provide the conditions to double the brightness and intensity of the beam from the PSB, thus removing the first bottleneck towards higher brightness for the LHC and simplifying operation. Moreover, this new linac constitutes an essential component of any of the envisaged LHC upgrade scenarios and could open the way to future extensions of the CERN accelerator complex towards higher performance. This Technical Design Report presents a detailed technical overview of the Linac4 design as it stands at end 2006

Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress

Plants Modify Biological Processes to Ensure Survival following Carbon Depletion: A Lolium perenne Model

BACKGROUND: Plants, due to their immobility, have evolved mechanisms allowing them to adapt to multiple environmental and management conditions. Short-term undesirable conditions (e.g. moisture deficit, cold temperatures) generally reduce photosynthetic carbon supply while increasing soluble carbohydrate accumulation. It is not known, however, what strategies plants may use in the long-term to adapt to situations resulting in net carbon depletion (i.e. reduced photosynthetic carbon supply and carbohydrate accumulation). In addition, many transcriptomic experiments have typically been undertaken under laboratory conditions; therefore, long-term acclimation strategies that plants use in natural environments are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Perennial ryegrass (Lolium perenne L.) was used as a model plant to define whether plants adapt to repetitive carbon depletion and to further elucidate their long-term acclimation mechanisms. Transcriptome changes in both lamina and stubble tissues of field-grown plants with depleted carbon reserves were characterised using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR data for select key genes indicated that plants reduced fructan degradation, and increased photosynthesis and fructan synthesis capacities following carbon depletion. This acclimatory response was not sufficient to prevent a reduction (P<0.001) in net biomass accumulation, but ensured that the plant survived. CONCLUSIONS: Adaptations of plants with depleted carbon reserves resulted in reduced post-defoliation carbon mobilization and earlier replenishment of carbon reserves, thereby ensuring survival and continued growth. These findings will help pave the way to improve plant biomass production, for either grazing livestock or biofuel purposes

Photocatalytic Nanolithography of Self-Assembled Monolayers and Proteins

Self-assembled monolayers of alkylthiolates on gold and alkylsilanes on silicon dioxide have been patterned photocatalytically on sub-100 nm length-scales using both apertured near-field and apertureless methods. Apertured lithography was carried out by means of an argon ion laser (364 nm) coupled to cantilever-type near-field probes with a thin film of titania deposited over the aperture. Apertureless lithography was carried out with a helium–cadmium laser (325 nm) to excite titanium-coated, contact-mode atomic force microscope (AFM) probes. This latter approach is readily implementable on any commercial AFM system. Photodegradation occurred in both cases through the localized photocatalytic degradation of the monolayer. For alkanethiols, degradation of one thiol exposed the bare substrate, enabling refunctionalization of the bare gold by a second, contrasting thiol. For alkylsilanes, degradation of the adsorbate molecule provided a facile means for protein patterning. Lines were written in a protein-resistant film formed by the adsorption of oligo(ethylene glycol)-functionalized trichlorosilanes on glass, leading to the formation of sub-100 nm adhesive, aldehyde-functionalized regions. These were derivatized with aminobutylnitrilotriacetic acid, and complexed with Ni2+, enabling the binding of histidine-labeled green fluorescent protein, which yielded bright fluorescence from 70-nm-wide lines that could be imaged clearly in a confocal microscope