Genetic engineering of baker’s and wine yeasts using formaldehyde hyperresistance-mediating plasmids

Yeast multi-copy vectors carrying the for maldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker’s and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use. Key words: · Yeast · Transformation · Hyperresistance to formaldehyd

Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene’s influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT–PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2{Delta}, rlf2{Delta} and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2{Delta} pso4-1 and rlf2{Delta} pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae

Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire

Idiosyncratic adverse drug reactions are unpredictable, dose independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkage between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells that required HLA-B*57:01 molecules for their function. However, the mechanism by which abacavir induces this pathologic T cell response remains unclear. Here we show that abacavir can bind within the F-pocket of the peptide-binding groove of HLA-B*57:01 thereby altering its specificity. This supports a novel explanation for HLA-linked idiosyncratic adverse drug reactions; namely that drugs can alter the repertoire of self-peptides presented to T cells thus causing the equivalent of an alloreactive T cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir, and that were recognized by T cells of hypersensitive patients. The assays we have established can be applied to test additional compounds with suspected HLA linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA linked hypersensitivities as well as guide the development of safer drugs

Sustainability appraisal: Jack of all trades, master of none?

Sustainable development is a commonly quoted goal for decision making and supports a large number of other discourses. Sustainability appraisal has a stated goal of supporting decision making for sustainable development. We suggest that the inherent flexibility of sustainability appraisal facilitates outcomes that often do not adhere to the three goals enshrined in most definitions of sustainable development: economic growth, environmental protection and enhancement, and the wellbeing of the human population. Current practice is for sustainable development to be disenfranchised through the interpretation of sustainability, whereby the best alternative is good enough even when unsustainable. Practitioners must carefully and transparently review the frameworks applied during sustainability appraisal to ensure that outcomes will meet the three goals, rather than focusing on a discourse that emphasises one or more goals at the expense of the other(s)

Moving from “superstar” cities to “ordinary” cities to reclaim middle-class status: gentrification-induced inter-metropolitan migration

A national reshuffling of the United States population is in progress, as ever more people adjust to the reality of excessively expensive urban housing markets. We unpack the out-migration of middle-class people from large, high-growth cities propelled by gentrification pressures, into one lower-cost metropolitan area in North Carolina: Raleigh-Durham-Chapel Hill (“the Triangle”), considering the multiple impacts of migrants on the region. We refine concepts that are central to the gentrification scholarship, namely who is displaced and the geographic range of displacement. We find that migrants experience many benefits in their new location but have fears about the future of their new city. As the Triangle is growing, it is reproducing the high prices, environmental harm, and loss of a sense of place migrants experienced in their city of origin. This suggests that newly growing cities such as the Triangle are at risk of replicating the inequalities that plague superstar cities. Theoretically, we reflect on how a case study of an “ordinary” city (Robinson [2002]. Global and world cities: A view from off the map. International Journal of Urban and Regional Research, 26(3), 531–554.; Robinson [2006]. Ordinary Cities: Between Modernity and development. Psychology Press.) in the United States South, an underrepresented region in urban studies, can shed new light on urbanization in the United States

Caribbean Spiny Lobster Fishery Is Underpinned by Trophic Subsidies from Chemosynthetic Primary Production

Data files are deposited with Figshare and are available at: https://dx.doi.org/10.6084/ m9.figshare.4225334

The K2-138 System: A Near-resonant Chain of Five Sub-Neptune Planets Discovered by Citizen Scientists

K2-138 is a moderately bright (V = 12.2, K = 10.3) main-sequence K star observed in Campaign 12 of the NASA K2 mission. It hosts five small (1.6-3.3 R⊕) transiting planets in a compact architecture. The periods of the five planets are 2.35, 3.56, 5.40, 8.26, and 12.76 days, forming an unbroken chain of near 3:2 resonances. Although we do not detect the predicted 2-5 minute transit timing variations (TTVs) with the K2 timing precision, they may be observable by higher-cadence observations with, for example, Spitzer or CHEOPS. The planets are amenable to mass measurement by precision radial velocity measurements, and therefore K2-138 could represent a new benchmark system for comparing radial velocity and TTV masses. K2-138 is the first exoplanet discovery by citizen scientists participating in the Exoplanet Explorers project on the Zooniverse platform

Magnetic properties of ilmenite-hematite single crystals from the Ecstall pluton near Prince Rupert, British Columbia

Paleomagnetic studies of the 91 Ma Ecstall pluton and other Cretaceous plutons of British Columbia imply large northward tectonic movements (>2000 km) may have occurred during the tectonic evolution of western North America. However, more recent studies have shown that the eastern edge of the Ecstall pluton experienced considerable mineralogical changes as younger Eocene plutons, such as the ∼58 Ma Quottoon Pluton, were emplaced along its margins. We investigated changes in the rock magnetic properties associated with this reheating event by examining isolated grains of intergrown ilmenite and hematite, the primary paleomagnetic recorder in the Ecstall pluton. Measurements of hysteresis properties, low-temperature remanence, and room temperature isothermal remanent magnetization acquisition and observations from magnetic force microscopy and off-axis electron holography indicate that samples fall into three groups. The groups are defined by the presence of mineral microstructures that are related to distance from the Quotoon plutonic complex. The two groups closest to the Quottoon Pluton contain magnetite within hematite and ilmenite lamellae. Reheating of the Ecstall pluton led to an increase in coercivity and magnetization, as well as to development of mixed phase hysteresis. These results indicate that shallow paleomagnetic directions from the western Ecstall pluton are not affected by reheating and are therefore likely to record original field conditions at the time of pluton emplacement. In the absence of structural deformation, these shallow inclinations are consistent with large-scale northward translation suggested by the Baja–British Columbia hypothesis