Characterization of transposon insertion mutants in desulfovibrio vulgaris hilderborough by sequencing genomic DNA [abstract]

Abstract only availableTn5 transposon mutagenesis occurs by a mechanism in which a segment of DNA (transposon) encoded in a plasmid is inserted into genomic DNA (the target) by a conservative (cut-and-paste) mechanism. When the insertion position is in a coding sequence or regulatory region of DNA, the insertion results in a mutation. The plasmid pRL27 encodes a mini-Tn5 transposon, Tn5 transposase, and kanamycin resistance, (Metcalf, William W. et al, 2002 Arch Microbiol 178 :193-201) and was used to transform Desulfovibrio vulgaris Hildenborough by electroporation. Transposon insertion mutants were identified by their ability to grow in the presence of kanamycin. To identify the insertion sites of the transposons, in theory one should be able to sequence from the transposon into chromosomal DNA and identify the mutation site by comparison with the known genome. Unlike sequencing of plasmid DNA or PCR products, direct genomic sequencing has a limited success rate. Direct genomic sequencing is sensitive to DNA quality, interference of secondary DNA structures, salt concentration, and the availability of primer binding sites. Because of these difficulties, in our attempts to identify insertion sites of mini-Tn5, we examined template DNA quality as well as modifying sequencing reaction conditions. Our objective is to develop an effective, reliable method for sequencing genomic DNA to identify where transposon insertion sites have occurred in each mutant.Department of Energy Genomics: Genomes to Life Progra

Sub-shot-noise shadow sensing with quantum correlations

The quantised nature of the electromagnetic field sets the classical limit to the sensitivity of position measurements. However, techniques based on the properties of quantum states can be exploited to accurately measure the relative displacement of a physical object beyond this classical limit. In this work, we use a simple scheme based on the split-detection of quantum correlations to measure the position of a shadow at the single-photon light level, with a precision that exceeds the shot-noise limit. This result is obtained by analysing the correlated signals of bi-photon pairs, created in parametric downconversion and detected by an electron multiplying CCD (EMCCD) camera employed as a split-detector. By comparing the measured statistics of spatially anticorrelated and uncorrelated photons we were able to observe a significant noise reduction corresponding to an improvement in position sensitivity of up to 17% (0.8dB). Our straightforward approach to sub-shot-noise position measurement is compatible with conventional shadow-sensing techniques based on the split-detection of light-fields, and yields an improvement that scales favourably with the detector’s quantum efficiency

Heat-dried sclerotia of Sclerotinia sclerotiorum myceliogenically germinate in water and are able to infect Brassica napus

The phytopathogenic fungus Sclerotinia sclerotiorum forms dormant structures (termed sclerotia) that germinate myceliogenically under certain environmental conditions. During myceliogenic germination, sclerotia produce hyphae, which can infect leaves or stems of host plants directly from the ground; this is termed basal infection. This study determined which abiotic conditions were most important for promoting myceliogenic germination of sclerotia in vitro. A high sclerotium hydration level and low incubation temperature (158C) improved mycelial growth in the presence of a nutrient source. Sclerotia incubated without a nutrient source on moist sand, vigorously myceliogenically germinated most frequently (63%) when they had been previously imbibed and then conditioned at -20°C. By far the most consistent amount of vigorous myceliogenic germination (>75%) was produced when sclerotia were heat-dried before being submerged in water. The hyphae of these sclerotia were shown to infect and proliferate on leaves of intact Brassica napus plants. This research provides a better understanding of the abiotic conditions that are likely to increase the risk of basal infection by S. sclerotiorum

Rapid automated characterization of transposon insertion mutants in Desulfovibrio vulgaris Hildenborough by srnPCR [abstract]

Abstract only availableTn5 transposon mutagenesis occurs by a mechanism in which a segment of DNA (transposon) encoded in a plasmid is inserted into genomic DNA (the target) by a conservative (cut-and-paste) mechanism (Fig. 2). When the insertion position is in a coding sequence or regulatory region of DNA, the insertion results in a mutation. The plasmid pRL27 (a generous gift from Bill Metcalf) encodes a mini-Tn5 transposon, Tn5 transposase, and kanamycin resistance (neo), and was used to transform Desulfovibrio vulgaris Hildenborough by electroporation. Transposon insertion mutants were identified by their ability to grow in the presence of kanamycin. To locate the insertion site of the transposon, in theory, one should be able to directly sequence from the transposon into chromosomal DNA (Fig. 3.1) and identify the mutation site by comparison with the known genome BLAST. Unlike sequencing of plasmid DNA or PCR products, direct genomic sequencing has a limited success rate. Therefore, a method of enriching the transposon-flanking sequence is needed. Nested semi-random PCR (Fig. 3.2) is an efficient and cost effective enrichment method. Sequencing these enriched products allows us to identify the transposon insertion site. The factors that influence characterization success rate are: frequency and location of priming sites, reaction volume, and reaction conditions (annealing temperature, extension time, etc.). By varying these factors, we have developed an efficient and reliable method for characterizing transposon insertion mutants. Utilizing high-throughput robotics and nested semi-random PCR, we have generated single gene mutants that may provide valuable biological data.U.S. Department of Energy Genomes to Life gran

Quantum Position Measurement of a Shadow: Beating the Classical Limit

The precision with which the position of a shadow can be measured is classically limited by shot-noise. We achieve sub-shot-noise position sensitivity by jointly detecting correlated photons with a simple split-detector scheme

Advanced technologies for future ground-based, laser-interferometric gravitational wave detectors

We present a review of modern optical techniques being used and developed for the field of gravitational wave detection. We describe the current state-of-the-art of gravitational waves detector technologies with regard to optical layouts, suspensions and test masses. We discuss the dominant sources and noise in each of these subsystems and the developments that will help mitigate them for future generations of detectors. We very briefly summarise some of the novel astrophysics that will be possible with these upgraded detectors

The complete genome sequence of the phytopathogenic fungus Sclerotinia sclerotiorum reveals insights into the genome architecture of broad host range pathogens

Sclerotinia sclerotiorum is a phytopathogenic fungus with over 400 hosts including numerous economically important cultivated species. This contrasts many economically destructive pathogens that only exhibit a single or very few hosts. Many plant pathogens exhibit a “two-speed” genome. So described because their genomes contain alternating gene rich, repeat sparse and gene poor, repeat-rich regions. In fungi, the repeat-rich regions may be subjected to a process termed repeat-induced point mutation (RIP). Both repeat activity and RIP are thought to play a significant role in evolution of secreted virulence proteins, termed effectors. We present a complete genome sequence of S. sclerotiorum generated using Single Molecule Real-Time Sequencing technology with highly accurate annotations produced using an extensive RNA sequencing data set. We identified 70 effector candidates and have highlighted their in planta expression profiles. Furthermore, we characterized the genome architecture of S. sclerotiorum in comparison to plant pathogens that exhibit “two-speed” genomes. We show that there is a significant association between positions of secreted proteins and regions with a high RIP index in S. sclerotiorum but we did not detect a correlation between secreted protein proportion and GC content. Neither did we detect a negative correlation between CDS content and secreted protein proportion across the S. sclerotiorum genome. We conclude that S. sclerotiorum exhibits subtle signatures of enhanced mutation of secreted proteins in specific genomic compartments as a result of transposition and RIP activity. However, these signatures are not observable at the whole-genome scale

Content Disputes in Wikipedia Reflect Geopolitical Instability

Indicators that rank countries according socioeconomic measurements are important tools for regional development and political reform. Those currently in widespread use are sometimes criticized for a lack of reproducibility or the inability to compare values over time, necessitating simple, fast and systematic measures. Here, we applied the ‘guilt by association’ principle often used in biological networks to the information network within the online encyclopedia Wikipedia to create an indicator quantifying the degree to which pages linked to a country are disputed by contributors. The indicator correlates with metrics of governance, political or economic stability about as well as they correlate with each other, and though faster and simpler, it is remarkably stable over time despite constant changes in the underlying disputes. For some countries, changes over a four year period appear to correlate with world events related to conflicts or economic problems