Mutagenesis and gene cloning in Schizosaccharomyces pombe using nonhomologous plasmid integration and rescue

Genes are commonly cloned in yeasts and bacteria by plasmid complementation, where the introduction of the gene of interest into a host strain carrying a recessive mutation in that gene suppresses the host's mutant phenotype. However, a lack of low copy cloning vectors in the fission yeast Schizosaccharomyces pombe can complicate this approach especially when overexpression of one gene may suppress a defect in another gene or when overexpression of the desired gene is detrimental, if not lethal, to the cell. We describe here a method of identifying mutations in S. pombe that allows for the rapid and direct cloning of the defective gene. This involves the nonhomologous integration of a marked plasmid into the yeast genome and its subsequent rescue into Escherichia coli, so that DNA at the site of insertion is incorporated into the recovered plasmid. As two of three insertions obtained in this study occurred outside of the affected gene's open reading frame, this method should be applicable to cloning both essential genes and nonessential genes.     

Geological history and within-island diversity: a debris avalanche and the Tenerife lizard Gallotia galloti

Several processes have been described that could explain geographical variation and speciation within small islands, including fragmentation of populations through volcanic eruptions. Massive landslides, or debris avalanches, could cause similar effects. Here we analyse the potential impact of the 0.8 million-year-ago (Ma) Güimar valley debris avalanche on the phylogeography of the lizard Gallotia galloti on the Canary Island of Tenerife. Distributions of mitochondrial DNA lineages (based on cytochrome b sequences) were analysed on a 60-km southeastern coast transect centred on this area. Three main clades were detected, which can be divided into northern (one clade) and southern (two clades) groups that introgress across the valley. Maximum-likelihood estimates of migration rates (scaled for mutation rate) revealed highly asymmetric patterns, indicating that long-term gene flow into this region from both the northern and the southern populations greatly exceeded that in the opposite directions, consistent with recolonization of the area. The ancestral Tenerife node on the G. galloti tree is estimated at 0.80 Ma, matching closely with the geological estimate for the debris avalanche. Morphological variation (body dimensions and scalation) was also analysed and indicated a stepped cline in female scalation across the valley, although the patterns for male scalation and male and female body dimensions were not as clear. Together these findings provide support for the hypothesis that the debris avalanche has shaped the phylogeography of G. galloti and may even have been a primary cause of the within-island cladogenesis through population fragmentation and isolation. Current estimates of timing of island unification mean that the original hypothesis that within-island diversity is explained by the secondary contact of populations from the two ancient precursor islands of Teno and Anaga is less plausible for this and some other Tenerife species. Large-scale landslides have occurred on many volcanic islands, and so may have been instrumental in shaping within-island diversities.     

Specific in vitro activation of Ca,Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K)

The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.     

Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport

Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life-saving protein therapeutics more available to the world, the possibility of magistral or point-of-care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized “cell-free” protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA-approved cancer therapeutics have increased its potential for low-cost, single-batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small-batch, cell-free protein synthesis (CFPS) reactions through air-water foams. These “hydrofoam” reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell-lysis and production losses, hydrofoam CFPS reactions are “cell-free” and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.     

Invertebrate colonisation of the gravel substratum of an experimental recirculating channel

The development of an invertebrate community in a large scale, experimental stream system is described. Colonisation was chiefly by means of ovipositing adult insects with lesser components introduced with water and stones. A succession of taxa was recorded with large peaks of numbers in the spring and the autumn, comparable to those observed in studies on natural streams. Diversity of the fauna increased quickly at first and then more slowly, possibly because of the reduced availability of ovipositing adults later in the year. Even in the absence of drift and migration, recruitment of insect larvae, which naturally favour gravel substrata in running water, can be rapid and will produce a varied community in which Chironomidae and Ephemeroptera play an important role.     

A simple activity monitoring radio tag for fish

A radio tag is described which facilitates the long-term monitoring of activity events in fish. The tag is a simple peritoneal implant and thus requires no complicated surgery to deploy. A motion-sensing mercury switch interfaced to a microcontroller is used to modulate pulse rate and thus indicate periods of activity. The tag is being used to study the feeding and activity patterns of pike (Esox lucius L.) where laboratory and field trials have shown the system to be robust, long lasting and reliable.