Characterization of a temperature-sensitive DNA ligase from Escherichia coli

DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of Escherichia coli, including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the ligA251 mutation in E. coli GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 °C purified LigA251 has 20-fold lower ligation activity in vitro, and its activity is reduced further at 42 °C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase

Systematic reviews: guidance relevant for studies of older people

Systematic reviews and meta-analyses are increasingly common. This article aims to provide guidance for people conducting systematic reviews relevant to the healthcare of older people. An awareness of these issues will also help people reading systematic reviews to determine whether the results will influence their clinical practice. It is essential that systematic reviews are performed by a team which includes the required technical and clinical expertise. Those performing reviews for the first time should ensure they have appropriate training and support. They must be planned and performed in a transparent and methodologically robust way: guidelines are available. The protocol should be written—and if possible published—before starting the review. Geriatricians will be interested in a table of baseline characteristics, which will help to determine if the studied samples or populations are similar to their patients. Reviews of studies of older people should consider how they will manage issues such as different age cut-offs; non-specific presentations; multiple predictors and outcomes; potential biases and confounders. Systematic reviews and meta-analyses may provide evidence to improve older people's care, or determine where new evidence is required. Newer methodologies, such as meta-analyses of individual level data, network meta-analyses and umbrella reviews, and realist synthesis, may improve the reliability and clinical utility of systematic reviews

Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4

The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single-strand break was determined. Between 4 and 37 °C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50-1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3'-hydroxy group and DNA at the 5'-phosphate. Since this RNA-DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 °C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of 'DNA repair'

Microsatellites for the marsh fritillary butterfly: de novo transcriptome sequencing, and a comparison with amplified fragment length polymorphism (AFLP) markers.

Journal ArticleResearch Support, Non-U.S. Gov'tBACKGROUND: Until recently the isolation of microsatellite markers from Lepidoptera has proved troublesome, expensive and time-consuming. Following on from a previous study of Edith's checkerspot butterfly, Euphydryas editha, we developed novel microsatellite markers for the vulnerable marsh fritillary butterfly, E. aurinia. Our goal was to optimize the process in order to reduce both time and cost relative to prevailing techniques. This was accomplished by using a combination of previously developed techniques: in silico mining of a de novo assembled transcriptome sequence, and genotyping the microsatellites found there using an economic method of fluorescently labelling primers. PRINCIPAL FINDINGS: In total, we screened nine polymorphic microsatellite markers, two of which were previously published, and seven that were isolated de novo. These markers were able to amplify across geographically isolated populations throughout Continental Europe and the UK. Significant deviations from Hardy-Weinberg equilibrium were evident in some populations, most likely due to the presence of null alleles. However, we used an F(st) outlier approach to show that these markers are likely selectively neutral. Furthermore, using a set of 128 individuals from 11 populations, we demonstrate consistency in population differentiation estimates with previously developed amplified fragment length polymorphism (AFLP) markers (r = 0.68, p<0.001). SIGNIFICANCE: Rapid development of microsatellite markers for difficult taxa such as Lepidoptera, and concordant results with other putatively neutral molecular markers, demonstrate the potential of de novo transcriptional sequencing for future studies of population structure and gene flow that are desperately needed for declining species across fragmented landscapes.BBSRCOkinawa Institute for Science and Technology (OIST

First results from the Very Small Array -- I. Observational methods

The Very Small Array (VSA) is a synthesis telescope designed to image faint structures in the cosmic microwave background on degree and sub-degree angular scales. The VSA has key differences from other CMB interferometers with the result that different systematic errors are expected. We have tested the operation of the VSA with a variety of blank-field and calibrator observations and cross-checked its calibration scale against independent measurements. We find that systematic effects can be suppressed below the thermal noise level in long observations; the overall calibration accuracy of the flux density scale is 3.5 percent and is limited by the external absolute calibration scale.Comment: 9 pages, 10 figures, MNRAS in press (Minor revisions

Surfactants for heterogeneous processes in liquid or supercritical CO.sub.2

Heterogeneous polymer mixtures comprising a polymer in liquid or supercritical CO.sub.2 are stabilized by employing a poly(propylene oxide) or poly(butylene oxide) based surfactant. These surfactants are especially well suited for stabilizing heterogeneous polymer mixtures formed by micronizing techniques as well as by precipitation of a compressed fluid antisolvent applications.Board of Regents, University of Texas Syste

Pyrosequencing the transcriptome of the greenhouse whitefly, Trialeurodes vaporariorum reveals multiple transcripts encoding insecticide targets and detoxifying enzymes.

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tBACKGROUND: The whitefly Trialeurodes vaporariorum is an economically important crop pest in temperate regions that has developed resistance to most classes of insecticides. However, the molecular mechanisms underlying resistance have not been characterised and, to date, progress has been hampered by a lack of nucleotide sequence data for this species. Here, we use pyrosequencing on the Roche 454-FLX platform to produce a substantial and annotated EST dataset. This 'unigene set' will form a critical reference point for quantitation of over-expressed messages via digital transcriptomics. RESULTS: Pyrosequencing produced around a million sequencing reads that assembled into 54,748 contigs, with an average length of 965 bp, representing a dramatic expansion of existing cDNA sequences available for T. vaporariorum (only 43 entries in GenBank at the time of this publication). BLAST searching of non-redundant databases returned 20,333 significant matches and those gene families potentially encoding gene products involved in insecticide resistance were manually curated and annotated. These include, enzymes potentially involved in the detoxification of xenobiotics and those encoding the targets of the major chemical classes of insecticides. A total of 57 P450s, 17 GSTs and 27 CCEs were identified along with 30 contigs encoding the target proteins of six different insecticide classes. CONCLUSION: Here, we have developed new transcriptomic resources for T. vaporariorum. These include a substantial and annotated EST dataset that will serve the community studying this important crop pest and will elucidate further the molecular mechanisms underlying insecticide resistance.CASE PhD studentship BBSRCBayer CropScienceRothamsted Researc

Sustained postsynaptic kainate receptor activation downregulates AMPA receptor surface expression and induces hippocampal LTD

It is well established that long-term depression (LTD) can be initiated by either NMDA or mGluR activation. Here we report that sustained activation of GluK2 subunit-containing kainate receptors (KARs) leads to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) endocytosis and induces LTD of AMPARs (KAR-LTD(AMPAR)) in hippocampal neurons. The KAR-evoked loss of surface AMPARs is blocked by the ionotropic KAR inhibitor UBP 310 indicating that KAR-LTD(AMPAR) requires KAR channel activity. Interestingly, however, blockade of PKC or PKA also reduces GluA2 surface expression and occludes the effect of KAR activation. In acute hippocampal slices, kainate application caused a significant loss of GluA2-containing AMPARs from synapses and long-lasting depression of AMPAR excitatory postsynaptic currents in CA1. These data, together with our previously reported KAR-LTP(AMPAR), demonstrate that KARs can bidirectionally regulate synaptic AMPARs and synaptic plasticity via different signaling pathways