The P-Loop Domain of Yeast Clp1 Mediates Interactions Between CF IA and CPF Factors in Pre-mRNA 3′ End Formation

Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3′ end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5′ RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3′ end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3′ end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3′ endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3′ end formation

Spatial variation of trace metals within intertidal beds of native mussels (Mytilus edulis) and non-native Pacific oysters (Crassostrea gigas): implications for the food web?

Abstract Pollution is of increasing concern within coastal regions and the prevalence of invasive species is also rising. Yet the impact of invasive species on the distribution and potential trophic transfer of metals has rarely been examined. Within European intertidal areas, the non-native Pacific oyster (Crassostrea gigas) is becoming established, forming reefs and displacing beds of the native blue mussel (Mytilus edulis). The main hypothesis tested is that the spatial pattern of metal accumulation within intertidal habitats will change should the abundance and distribution of C. gigas continue to increase. A comparative analysis of trace metal content (cadmium, lead, copper and zinc) in both species was carried out at four shores in south-east England. Metal concentrations in bivalve and sediment samples were determined after acid digestion by inductively coupled plasma-optical emission spectrometry. Although results showed variation in the quantities of zinc, copper and lead (mg m-2) in the two bivalve species, differences in shell thickness are also likely to influence the feeding behaviour of predators and intake of metals. The availability and potential for trophic transfer of metals within the coastal food web, should Pacific oysters transform intertidal habitats, is discussed

Measurement of immunoglobulin A in saliva by particle-enhanced nephelometric immunoassay: sample collection, limits of quantitation, precision, stability and reference range

Background: Total immunoglobulin A in saliva (s-IgA) is normally assayed using an enzyme-linked immunosorbent assay. We have investigated methodological issues relating to the use of particle-enhanced nephelometric immunoassay (PENIA)to measure s-IgA in whole unstimulated saliva and determine its reference range.Methods: Whole unstimulated resting saliva was collected to determine sample stability (temperature, time, effect of a protease inhibitor), limit of quantitation (LOQ), assay precision and analytical variation. The reference range for 134 healthy adults was determined.Results: Linearity was excellent (4–10.3 mg L21, P, 0.001; R2 ¼ 0.997) and without significant bias (mean of 20.7%). The lowest intra- and inter-analytical coefficients of variation were 1.8% and 7.5% and LOQ was 1.4 mg L21. The concentration of s-IgA is stable at room temperature for up to 6 h, at 48C for 48 h, at 248C for two weeks and at 2808C for up to 1.3 yr. There is no evidence that a protease inhibitor increases the stability or that repeated freeze–thawing cycles degrade sample quality. The reference ranges for s-IgA concentration, s-IgA secretion, s-IgA:albumin and s-IgA:osmolality were 15.9–414.5 mg L21, 7.2–234.9 mg min21, 0.4–19 and 0.6–8.9, respectively.Conclusion: Automated PENIA assay of s-IgA is precise and accurate. High stability of collected saliva samples and the ease and speed of the assay make this an ideal method for use in athletic and military training situations. The convenience of measuring albumin and IgA on the same analytical platform adds to the practicability of the test

Norm enforcement as supervisory control

In this paper, we study normative multi-agent systems from a supervisory control theory perspective. Concretely, we show how to model three well-known types of norm enforcement mechanisms by adopting well-studied supervisory control theory techniques for discrete event systems. Doing so provides a semantics for normative multi-agent systems rooted in formal languages and the ability to automatically synthesize SCT-based norm enforcement mechanisms for special, but still fairly expressive, type of systems and properties