Origin-Dependent Inverted-Repeat Amplification: Tests of a Model for Inverted DNA Amplification

DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.     

Morphology and life cycle of a new loriciferan from the Atlantic coast of Florida with an emended diagnosis and life cycle of Nanaloricidae (Loricifera)

Abstract. A new interstitial loriciferan, Nanaloricus gwenae sp. nov., is described from coarse-sand and shell-hash habitats ( Amphioxus sand), at 15–17 m depth, 6–7 miles off the coast of Fort Pierce, FL, USA. The new species is very closely related to the type species Nanaloricus mysticus found off the coast of Roscoff, France in nearly the same kind of sediment ( Dentalium sand). All life stages (Higgins larva, postlarva, and adult) in the life cycle of the new species were found through the 10-year investigation, leading to the conclusion that all species of Nanaloricidae have only a sexual reproductive cycle. The adult of the new species can be distinguished by the different body shape, the lorical spikes and sculpture, and the shape of the scalids. The postlarva has stronger longitudinal ridges on the lorical plates and the Higgins larva has toes with smaller mucrones than those of N. mysticus . An emended definition of the Nanaloricidae is provided based on the new formula of the number of rows and shape of the scalids on the introvert. The associated meiofauna found in the subtidal sand or shell hash is both abundant and diverse. Tardigrades, gastrotrichs, and kinorhynchs are among the most common associates. N . gwenae sp. nov. is represented by only eight specimens collected during a 10-year period of sampling this habitat.     

Sequential shrinkage and swelling underlie P2X7-stimulated lymphocyte phosphatidylserine exposure and death

Patterns of change in cell volume and plasma membrane phospholipid distribution during cell death are regarded as diagnostic means of distinguishing apoptosis from necrosis, the former being associated with cell shrinkage and early phosphatidylserine (PS) exposure, whereas necrosis is associated with cell swelling and consequent lysis. We demonstrate that cell volume regulation during lymphocyte death stimulated via the purinergic receptor P2X7 is distinct from both. Within seconds of stimulation, murine lymphocytes undergo rapid shrinkage concomitant with, but also required for, PS exposure. However, within 2 min shrinkage is reversed and swelling ensues ending in cell rupture. P2X7-induced shrinkage and PS translocation depend upon K+ efflux via KCa3.1, but use a pathway of Cl- efflux distinct from that previously implicated in apoptosis. Thus, P2X7 stimulation activates a novel pathway of cell death that does not conform to those conventionally associated with apoptosis and necrosis. The mixed apoptotic/necrotic phenotype of P2X7-stimulated cells is consistent with a potential role for this death pathway in lupus disease.     

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.     

Modelling changes in species’ abundance in response to projected climate change

Aim Existing climate envelope models give an indication of broad scale shifts in distribution, but do not specifically provide information on likely future population changes useful for conservation prioritization and planning. We demonstrate how these techniques can be developed to model likely future changes in absolute density and population size as a result of climate change. Location Great Britain. Methods Generalized linear models were used to model breeding densities of two northerly‐ and two southerly‐distributed bird species as a function of climate and land use. Models were built using count data from extensive national bird monitoring data and incorporated detectability to estimate absolute abundance. Projections of likely future changes in the distribution and abundance of these species were made by applying these models to projections of future climate change under two emissions scenarios. Results Models described current spatial variation in abundance for three of the four species and produced modelled current estimates of national populations that were similar to previously published estimates for all species. Climate change was projected to result in national population declines in the two northerly‐distributed species, with declines for Eurasian curlew Numenius arquata projected to be particularly severe. Conversely, the abundances of the two southerly distributed species were projected to increase nationally. Projected maps of future abundance may be used to identify priority areas for the future conservation of each species. Main conclusions The analytical methods provide a framework to make projections of impacts of climate change on species abundance, rather than simply projected range changes. Outputs may be summarized at any spatial scale, providing information to inform future conservation planning at national, regional and local scales. Results suggest that as a consequence of climate change, northerly distributed bird species in Great Britain are likely to become an increasingly high conservation priority within the UK.