Identification of Nucleases and Phosphatases by Direct Biochemical Screen of the Saccharomyces cerevisiae Proteome

The availability of yeast strain collections expressing individually tagged proteins to facilitate one-step purification provides a powerful approach to identify proteins with particular biochemical activities. To identify novel exo- and endo-nucleases that might function in DNA repair, we undertook a proteomic screen making use of the movable ORF (MORF) library of yeast expression plasmids. This library consists of 5,854 yeast strains each expressing a unique yeast ORF fused to a tripartite tag consisting of His6, an HA epitope, a protease 3C cleavage site, and the IgG-binding domain (ZZ) from protein A, under the control of the GAL1 promoter for inducible expression. Pools of proteins were partially purified on IgG sepharose and tested for nuclease activity using three different radiolabeled DNA substrates. Several known nucleases and phosphatases were identified, as well as two new members of the histidine phosphatase superfamily, which includes phosphoglycerate mutases and phosphatases. Subsequent characterization revealed YDR051c/Det1 to be an acid phosphatase with broad substrate specificity, whereas YOR283w has a broad pH range and hydrolyzes hydrophilic phosphorylated substrates. Although no new nuclease activities were identified from this screen, we did find phosphatase activity associated with a protein of unknown function, YOR283w, and with the recently characterized protein Det1. This knowledge should guide further genetic and biochemical characterization of these proteins

Sorting the Assemblies of Unsymmetrically Covalently Functionalized Mn-Anderson Polyoxometalate Clusters with Mass Spectrometry

The unsymmetrical Mn-Anderson polyoxometalate cluster, [N(C4H9)(4)](3)[MnMo6O18(C4H6O3NO2)(C4H6O3NH2)] (1; N(C4H9)(4)(+) = TBA(+)), has been prepared and characterized by X-ray crystallography and electrospray ionization mass spectrometry (ESI-MS). Covalent functionalization of compound 1 leads to the controlled assemblies of unsymmetrical alkoxopolyoxometalate clusters of compounds 2-5, which can be directly observed in solution as revealed by ESI-MS studies

A degree is not enough: a quantitative study of aspects of pre-service science teachers' chemistry content knowledge

Aspects of chemistry content knowledge held by 265 UK-based pre-service teachers (PSTs) were probed using 28 diagnostic questions in five chemistry concept areas, Particle theory and changes of state, Mass conservation (taught to 11–14-year-olds), and Chemical bonding, Mole calculations and Combustion reactions (taught to 14–16-year-olds). Data were collected over six years from academically able science graduates starting a full-time, university-based teacher education programme of one academic year duration. PSTs in three sub-cohorts (‘chemists', ‘physicists' and ‘biologists' on the basis of their undergraduate degrees) demonstrated similar levels of content knowledge (CK) for Particle theory and changes of state and Mass conservation. Biologists demonstrated statistically significantly weaker understanding than chemists and physicists in Chemical bonding, Mole calculations and Combustion reactions. Forty-four ‘triads' each comprising one chemist, physicist and biologist, matched by academic and personal backgrounds, showed that chemists outperformed biologists and physicists in Chemical bonding and Combustion reactions. The findings suggest that non-chemists' CK is insufficient for teaching these chemistry concepts in high schools, despite their possession of ‘good' Bachelor of Science degrees. These data have implications for science teacher education, including how best to prepare science graduates from diverse backgrounds for teaching specialist science subjects to 11–16-year-olds