No abstract

SummaryCatalysis of DNA recombination by Tn 3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites ( res ). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli . Some single mutants can resolve a res ¥ ¥ ¥ ¥ site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I ¥ ¥ ¥ ¥ site I plasmid. Site I ¥ ¥ ¥ ¥ site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3 ¢ ¢ ¢ ¢ interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.

Xer site-specific recombination is required for the stable inheritance of multicopy plasmids and the normal segregation of the bacterial chromosome in Escherichia coli. Two related recombinases and two accessory proteins are essential for Xer-mediated recombination at cer, a recombination site in the plasmid ColE1. The accessory proteins, ArgR and PepA, function in ensuring that the Xer recombination reaction acts exclusively intramolecularly, converting plasmid dimers into monomers and not vice versa. PepA is an amino-exopeptidase, but its molecular role in the Xer recombination mechanism is unclear. Here we show that a mutation directed at the presumptive active site of PepA creates a protein with no detectable peptidase activity in vitro or in vivo, but which still functions normally in Xer site-specific recombination at cer.

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.

The resolvases of the bacterial transposons Tn3 and γδ are closely related DNA site-specific recombinases and founder members of the serine recombinase family (Rowland and Stark, 2005; Stark, 2014). They function to resolve cointegrate intermediates of replicative transposition (Grindley, 2002), and are characterized by stringent topological selectivity: recombination (resolution) is licensed only when two recombination sites (res) are oriented in direct repeat (head to tail) within a supercoiled DNA molecule. Each res has three binding sites for resolvase dimers (Figure 1a); one of these (site I, the crossover site) is centred on the bonds that are broken and rejoined during recombination. In a productive reaction, the two res sites and six bound resolvase dimers assemble into an interwound synapse

Nuclear and mtDNA sequences from selected short-looped terebratuloid (terebratulacean) articulate brachiopods yield congruent and genetically independent phylogenetic reconstructions by parsimony, neighbour-joining and maximum likelihood methods, suggesting that both sources of data are reliable guides to brachiopod species phylogeny. The present-day genealogical relationships and geographical distributions of the tested terebratuloid brachiopods are consistent with a tethyan dispersal and subsequent radiation. Concordance of nuclear and mitochondrial gene phylogenies reinforces previous indications that articulate brachiopods, inarticulate brachiopods, phoronids and ectoprocts cluster with other organisms generally regarded as protostomes. Since ontogeny and morphology in brachiopods, ectoprocts and phoronids depart in important respects from those features supposedly diagnostic of protostomes, this demonstrates that the operational de¢nition of protostomy by the usual ontological characters must be misleading or unreliable. New, molecular, operational de¢nitions are proposed to replace the traditional criteria for the recognition of protostomes and deuterostomes, and the clade-based terms Protostomozoa' and`Deuterostomozoa' are proposed to replace the existing terms`Protostomia' and Deuterostomia'.

Gestational diabetes is associated with increased AF insulin at 14-20 weeks, suggesting augmentation of fetal insulin production in the early fetal period in at least some cases of gestational diabetes.

The findings of this study respond to a gap in the literature in Ireland on the experiences of healthcare professionals who have implemented ICPs. Key findings of the study are the perception that the doctor is pivotal in driving the implementation of ICPs, yet the doctor was not always interested in this responsibility.