DNA sequencing of four bases using three lanes.

A three-lane DNA sequencing strategy is described which is based on redundant binary coding principles from communications theory. Three-lane sequencing is an efficient, accurate, and flexible strategy, suitable for large-scale automated DNA sequencing. Communications theory and algebraic coding principles can also be applied to sequencing other informational macromolecules.     

A regional impact assessment of climate and land-use change on alpine vegetation

Aim Assessing potential response of alpine plant species distribution to different future climatic and land‐use scenarios. Location Four mountain ranges totalling 150 km² in the north‐eastern Calcareous Alps of Austria. Methods Ordinal regression models of eighty‐five alpine plant species based on environmental constraints and land use determining their abundance. Site conditions are simulated spatially using a GIS, a Digital Terrain Model, meteorological station data and existing maps. Additionally, historical records were investigated to derive data on time spans since pastures were abandoned. This was then used to assess land‐use impacts on vegetation patterns in combination with climatic changes. Results A regionalized GCM scenario for 2050 (+ 0.65 °C, ?30 mm August precipitation) will only lead to local loss of potential habitat for alpine plant species. More profound changes (+ 2 °C, ?30 mm August precipitation; + 2 °C, ?60 mm August precipitation) however, will bring about a severe contraction of the alpine, non‐forest zone, because of range expansion of the treeline conifer Pinus mugo Turra and many alpine species will loose major parts of their habitat. Precipitation change significantly influences predicted future habitat patterns, mostly by enhancing the general trend. Maintenance of summer pastures facilitates the persistence of alpine plant species by providing refuges, but existing pastures are too small in the area to effectively prevent the regional extinction risk of alpine plant species. Main conclusions The results support earlier hypotheses that alpine plant species on mountain ranges with restricted habitat availability above the treeline will experience severe fragmentation and habitat loss, but only if the mean annual temperature increases by 2 °C or more. Even in temperate alpine regions it is important to consider precipitation in addition to temperature when climate impacts are to be assessed. The maintenance of large summer farms may contribute to preventing the expected loss of non‐forest habitats for alpine plant species. Conceptual and technical shortcomings of static equilibrium modelling limit the mechanistic understanding of the processes involved.     

A novel antibiotic free plasmid selection system: advances in safe and efficient DNA therapy

The presence of antibiotic resistance genes in the delivered plasmids is one of the drawbacks of modern gene therapy and DNA vaccine applications. Here, we describe a strategy that allows for plasmid selection in bacterial hosts, without the requirement of any selection marker. Several bacterial strains were modified, so that the plasmid's replicational inhibitor RNA I could suppress the translation of a growth essential gene by RNA-RNA antisense reaction. An essential gene (murA) was modified such that a repressor protein (tetR) would hamper its expression. Only in the presence of plasmid and, hence, RNA I, was tetR turned down and murA expressed. Different commercially available plasmids could be selected by various modified Escherichia coli strains. We further designed a minimalistic plasmid devoid of any selection marker. All of the clones (n=6) examined, when the modified strain JM109-murselect was used for selection, contained plasmids. Thus, we have designed bacterial host strains that for the first time serve to select and maintain plasmids without the use of any selection marker or other additional sequence on the plasmid. Consequently, such plasmids may not only be safer, but due to their decreased size, advantages for the manufacturer and higher transfection efficiencies are anticipated.     

Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome‐integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon‐limited microbioreactor cultivations with yields of 0–7.4 mg Fab per gram of cell dry mass. Antigen‐binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid‐free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80–300% of plasmid‐based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.     

A fine-scaled predictive model for changes in species distribution patterns of high mountain plants induced by climate warming

Induced by global warming, mountain plant species are migrating upwards. Species inhabiting the nival zone of today are threatened by competitors which move from the alpine zone towards the summits. The manner in which species move depends on their abilities to cope with microtopographical situations. We present a spatially explicit predictive model which draws scenarios of future species distribution patterns at a typical high mountain of the European Alps. The altitudinal temperature gradient is examined. Based on the lapse rate and on definitions of topographical niches of species, a +1 °C- and a +2 °C-warming scenario are modelled using a fine-scaled digital elevation model. Nival species will lose area and become restricted to specific topographical situations. Alpine and subnival grassland species are predicted to expand their area, mainly along stable surface situations. Whether the migration will take place as a filling or a moving process is specific to the particular species. Overall, biodiversity is apparently not threatened on the decadal scale. In special cases, however, genetic losses are likely both on a local and on a regional scale.     

Expanding baculovirus surface display. Modification of the native coat protein gp64 of Autographa californica NPV.

To create a tool for eukaryotic surface display, this approach is aimed at demonstrating a direct modification of the native envelope protein gp64 of Autographa californica NPV without disturbing viral infectivity. Short affinity-tag peptides, the biotin mimic streptagII, and the gp41 amino-acid motif ELDKWA of HIV-1, specific for the human monoclonal antibody 2F5, were engineered into the baculovirus major coat protein gp64 and presented on the viral surface. Two different streptag peptides were inserted at the naturally occurring NotI site at amino-acid 278 of gp64. Additionally, the ten-amino-acid peptide GG-ELDKWA-GG, containing the epitope of mAb 2F5, was introduced into gp64 envelope protein at the same position. In all cases we were able to propagate viable virus-achieving infectious titers in the range of wild-type AcMNPV. Streptag and ELDKWA-epitope surface localization on purified virus particles was demonstrated by flow cytometry and Western blot analysis. We could also show selective retention of mutant viruses by specific interaction between chimeric virions and their target counterparts, recognizing the epitope or the streptag peptide in the viral envelope. These data provide evidence that altering the surface properties of the baculovirus virion could be of value in improving baculovirus display technology and developing new applications.