CRISPR-Cas immunity and mobile DNA: a new superfamily of DNA transposons encoding a Cas1 endonuclease

Mobile genetic elements such as DNA transposons are a feature of most genomes. The existence of novel DNA transposons can be inferred when whole genome sequencing reveals the presence of hallmarks of mobile elements such as terminal inverted repeats (TIRs) flanked by target site duplications (TSDs). A recent report describes a new superfamily of DNA transposons in the genomes of a few bacteria and archaea that possess TIRs and TSDs, and encode several conserved genes including a cas1 endonuclease gene, previously associated only with CRISPR-Cas adaptive immune systems. The data strongly suggests that these elements, designated ‘casposons’, are likely to be bona fide DNA transposons and that their Cas1 nucleases act as transposases and are possibly still active

Reconstitution of a functional IS608 single-strand transpososome: role of non-canonical base pairing

Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5′ to the foot of each IP, guide (G) sequences, using a network of canonical (‘Watson–Crick’) base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions

Polymerization and nucleic acid-binding properties of human L1 ORF1 protein

The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference—the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival

Sights and insights: Vocational outdoor students’ learning

Outdoor leader and adventure sport education in the United Kingdom has been characterized by an over-emphasis on technical skills at the expense of equally important, but often marginalized intra- and inter-personal skills necessary for contemporary outdoor employment. This study examined the lived experience of vocational outdoor students in order, firstly, to identify what was learned about the workplace through using reflective practice and, secondly, what was learned about reflective practice through this experience. The study used a purposive sample of students (n=15) who were invited to maintain reflective journals during summer work experience, and this was followed up with semi-structured interviews. Manual Interpretative Phenomenological Analysis (IPA) revealed that in the workplace setting students used reflective practice to understand and develop technical proficiency, support awareness of the value of theory, and acted as a platform to express emergent concepts of ‘professionalism’. Lessons about reflective practice emphasized its value in social settings, acknowledging different ways of reflection, and understanding and managing professional life beyond graduation

Potential impact of invasive alien species on ecosystem services provided by a tropical forested ecosystem: a case study from Montserrat

Local stakeholders at the important but vulnerable Centre Hills on Montserrat consider that the continued presence of feral livestock (particularly goats and pigs) may lead to widespread replacement of the reserve’s native vegetation by invasive alien trees (Java plum and guava), and consequent negative impacts on native animal species. Since 2009, a hunting programme to control the feral livestock has been in operation. However long-term funding is not assured. Here, we estimate the effect of feral livestock control on ecosystem services provided by the forest to evaluate whether the biodiversity conservation rationale for continuation of the control programme is supported by an economic case. A new practical tool (Toolkit for Ecosystem Service Site-based Assessment) was employed to measure and compare ecosystem service provision between two states of the reserve (i.e. presence and absence of feral livestock control) to estimate the net consequences of the hunting programme on ecosystem services provided by the forest. Based on this we estimate that cessation of feral livestock management would substantially reduce the net benefits provided by the site, including a 46 % reduction in nature-based tourism (from $419,000 to$228,000) and 36 % reduction in harvested wild meat (from $205,000 to$132,000). The overall net benefit generated from annual ecosystem service flows associated with livestock control in thereserve, minus the management cost, was $214,000 per year. We conclude that continued feral livestock control is important for maintaining the current level of ecosystem services provided by the reserve

Impact of Optimized Breastfeeding on the Costs of Necrotizing Enterocolitis in Extremely Low Birthweight Infants

To estimate risk of NEC for ELBW infants as a function of preterm formula and maternal milk (MM) intake and calculate the impact of suboptimal feeding on NEC incidence and costs

Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding

Abstract The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end