Simultaneous transformation of Escherichia coli by pairs of compatible and incompatible plasmid DNA molecules.

Summary This paper describes experiments involving simultaneous transformation of Escherichia coli by DNA of two species of multicopy plasmid in order to study competence and DNA uptake in this organism. In transformation mixtures where separate species of plasmid DNA were present in equal amounts, selection for a single plasmid gave doseresponse curves with slopes of 1. These results indicate that uptake of a single molecule of plasmid DNA is sufficient to produce one transformant. Simultaneous selection for markers on both plamsids gave a dose-response curve with a slope of 2. The total numbers of transformants obtained in single or double transformation experiments at saturating DNA concentrations were the same, and represented the maximum number of transformable cells. However, the absolute frequency of double transformants was reduced 4–6 fold relative to frequencies obtained with single markers. Similar results were obtained using pairs of compatible or incompatible plasmids in rec ⁺ or recA strains. These findings may be explained either on the basis of interference between competing plasmid molecules for uptake or establishment or by assuming that competent cells of Escherichia coli vary in their ability to incorporate more than one plasmid DNA molecule. Our results are consistent with an interpretation where more than 80% of the transformable cells are only capable of establishing one plasmid moiecule.     

Metabolism and activation of benzo[a]pyrene by mouse and rat skin in short-term organ culture and in vivo

The metabolic activation of BP was examined in mouse and rat skin in vivo and in short-term organ culture. In mouse skin, larger quantities of ether- and water-soluble metabolites were formed and more BP became bound covalently to DNA and protein than in rat skin. Qualitative differences in the formation of dihydrodiol metabolites and of BP-deoxyribonucleoside adducts between mouse and rat skin were also observed. Organ culture techniques may not provide a true model of metabolic activation in vivo because it was found that the covalent binding of BP to DNA and protein was reduced in skin maintained in culture despite an accumulation of dihydrodiol and other ether-soluble metabolites. In addition, the proportions of the syn- and anti-isomers of BP-7,8-diol 9,10-oxide involved in the formation of adducts with deoxyguanosine differed between skin treated in organ culture and in vivo.     

X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution.

The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.     

Detection and quantification of triazine herbicides using algal cell fluorescence

Summary A continuous-flow system is described which, by measuring fluorescence of the unicellular alga Chlorella, is capable of measuring concentrations of the triazine herbicide, simazine, as low as 60nM (approx 12g l^-1) within 5 minutes. Further developments are suggested to achieve the desired detection limit of 0.5nM. The use of such an instrument in environmental analysis is discussed.     

Analysis of developmentally homogeneous neural crest cell populations in vitro: I. Formation,morphology and differentiative behavior

When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters containing several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation.     

DNA and protein content of mouse sperm: Implications regarding sperm chromatin structure

A variety of biochemical and histochemical techniques have been used to compare the composition of chromatin in sperm nuclei isolated from the epididymides of five mouse strains. The DNA content was determined by phosphorus analysis, deoxyribose analysis, absorption spectroscopy at 260 nm, and cytomorphometry following gallocyanine chrome alum staining. All four methods indicate that the mouse sperm nucleus contains approx. 3.3 pg DNA and that the DNA content does not vary significantly among the strains tested. Three different techniques, quantitative amino acid analysis, absorption spectroscopy at 230 nm, and sperm head density analysis in cesium chloride, were used to determine the protein content. Sperm nuclei from each strain of mouse were found to have a protein to DNA ratio of 0.9 and a chromatin protein content of 3 pg/nucleus. Comparisons of the basic proteins by disc gel electrophoresis demonstrate that the sperm nuclei contain only protamine and lack significant levels of somatic histones or transition proteins. The sperm from each strain contained both mouse protamine variants and the relative distribution of the two proteins did not appear to differ among strains. Using this information, we have been able to draw certain conclusions regarding DNA-protamine interactions and the mode of DNA packaging in the sperm nucleus. The most important of these is that the DNA in the mouse sperm nucleus cannot be packaged in nucleosomes. The protamines in sperm chromatin do not function as structural proteins, providing a subunit core around which the DNA is wrapped, but appear to completely neutralize the phosphodiester backbone of the DNA molecule, thereby minimizing the repulsion between neighboring segments of DNA and allowing it to be condensed into a biochemically inactive particle of genetic information.     

Genomics in a changing arctic: critical questions await the molecular ecologist

Molecular ecology is poised to tackle a host of interesting questions in the coming years. The Arctic provides a unique and rapidly changing environment with a suite of emerging research needs that can be addressed through genetics and genomics. Here we highlight recent research on boreal and tundra ecosystems and put forth a series of questions related to plant and microbial responses to climate change that can benefit from technologies and analytical approaches contained within the molecular ecologist's toolbox. These questions include understanding (i) the mechanisms of plant acquisition and uptake of N in cold soils, (ii) how these processes are mediated by root traits, (iii) the role played by the plant microbiome in cycling C and nutrients within high‐latitude ecosystems and (iv) plant adaptation to extreme Arctic climates. We highlight how contributions can be made in these areas through studies that target model and nonmodel organisms and emphasize that the sequencing of the Populus and Salix genomes provides a valuable resource for scientific discoveries related to the plant microbiome and plant adaptation in the Arctic. Moreover, there exists an exciting role to play in model development, including incorporating genetic and evolutionary knowledge into ecosystem and Earth System Models. In this regard, the molecular ecologist provides a valuable perspective on plant genetics as a driver for community biodiversity, and how ecological and evolutionary forces govern community dynamics in a rapidly changing climate.     

A simple topographical model to predict Golden Eagle Aquila chrysaetos space use during dispersal

Many large raptors exploit or rely on anabatic and orographic winds which provide vertical lift, to supplement or provide the energy fuelling flight. Airspace is therefore a critical habitat for such large raptors and its use is subject to the underlying terrestrial topography, because particular topographical features are more likely to provide wind-energetic lift. Accordingly, ridges and/or ‘rugged topography’ are common preferred features in habitat use by large raptors. Our study aimed to provide a simple model of space use for a large raptor, the Golden Eagle Aquila chrysaetos, based on thousands of GPS telemetry records during juvenile dispersal of 92 birds tagged as nestlings between 2007 and 2016 across upland Scotland. Model development was based on the hypothesis that four topographical variables would be influential: slope, aspect, altitude and distance from ridge. The telemetry dataset was divided into training and two testing components. The first testing set was derived by a temporal split resulting in approximately equal sample size on records and some temporal overlap in individuals’ records with training data. The second testing set involved no individuals from the training set. Aspect was removed early in training model development because it was not influential. The model found that young Golden Eagles preferred, or used according to availability, space above slopes greater than 10°, at an altitude of ≥ 300 m, and within 300 m of a ridge. The test data were highly correlated with those from the training data in the model variables, and performance as regard to expected preferences from the model was improved in both test datasets, indicating the model was robust. Given the apparent universal nature of large raptor dependence on topography, that topography is relatively immutable according to time and use, and that topographical data are readily available, we commend our approach to other habitat preference studies of Golden Eagles and other large raptors elsewhere.     

Characterising the seasonal cycle of dissolved organic nitrogen using Cefas SmartBuoy high-resolution time-series samples from the southern North Sea

The Cefas SmartBuoy network provides a unique insight into the biogeochemical dynamics of the Northern European shelf seas, particularly the North Sea, through high-resolution automated offshore water sampling. We present total dissolved nitrogen and dissolved organic nitrogen (DON) from the Dowsing SmartBuoy site (53.531° N, 1.053° E) from January to October 2010, the first high resolution seasonal (winter-autumn) cycle of DON from the open North Sea. On top of a refractory background DON concentration of approximately 5 μM, a rapid increase in DON of a further ～5 μM is observed over the course of the spring bloom. This rapidly produced DON declines at an estimated net decay rate of between 0.6 and 1.8 μM month^?1. The slow decay suggests that the majority of the additional DON produced during the spring bloom is of semi-labile nature and has a lifetime of weeks to months. The dataset allows us to tightly constrain the budget for water column nitrogen over the winter, spring and summer of 2010 and clearly demonstrates the ‘sawtooth’ nature of the seasonal cycle of DON in the open North Sea, which has been impossible to resolve with a more traditional ship-based mode of operation. This work highlights the importance of autonomous sampling approaches in better understanding shelf sea biogeochemistry in the future.