517 research outputs found

    Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

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    Background: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 59 or 29 OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. Methodology/Principal Findings: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 59 phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A2.A1.A3.A4.A0.A6.A8.A10 and T2.T3.T4.T6<T1.T8.T10. Bridging T’s generally gave higher yield of ligated product than bridging A’s. ATP concentrations above 33 mM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (.90%) joined a correctly paired, or T:G wobble-paired, substrate on the 39 side of the ligation junction while discriminating approximately 100-fold against most mispaire

    Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

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    We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

    The Dictyostelium discoideum acaA Gene Is Transcribed from Alternative Promoters during Aggregation and Multicellular Development

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    Background: Extracellular cAMP is a key extracellular signaling molecule that regulates aggregation, cell differentiation and morphogenesis during multi-cellular development of the social amoeba Dictyostelium discoideum. This molecule is produced by three different adenylyl cyclases, encoded by the genes acaA, acrA and acgA, expressed at different stages of development and in different structures. Methodology/Principal Findings: This article describes the characterization of the promoter region of the acaA gene, showing that it is transcribed from three different alternative promoters. The distal promoter, promoter 1, is active during the aggregation process while the more proximal promoters are active in tip-organiser and posterior regions of the structures. A DNA fragment containing the three promoters drove expression to these same regions and similar results were obtained by in situ hybridization. Analyses of mRNA expression by quantitative RT-PCR with specific primers for each of the three transcripts also demonstrated their different temporal patterns of expression. Conclusions/Significance: The existence of an aggregation-specific promoter can be associated with the use of cAMP as chemo-attractant molecule, which is specific for some Dictyostelium species. Expression at late developmental stages indicates that adenylyl cyclase A might play a more important role in post-aggregative development than previously considered
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