Preparation and identification of partially fractionated Chinese hamster chromosomes.

Mitotic Chinese hamster chromosomes were isolated from synchronized, cultured M3-1 cells by hypotonic swelling followed by homogenization. The chromosomes were then fractionated by size by sucrose gradient sedimentation centrifugation. The resolution of the fractionation was determined by studying the morphology and Giemsa-banding patterns of the chromosomes found in each fraction. Fractions were obtained which were composed of as much as 50% of one chromosome type. Preparations such as these can be used for large scale physicochemical studies of single chromosome types.     

Plasmid- and strain-specific factors drive variation in ESBL-plasmid spread in vitro and in vivo

Horizontal gene transfer, mediated by conjugative plasmids, is a major driver of the global rise of antibiotic resistance. However, the relative contributions of factors that underlie the spread of plasmids and their roles in conjugation in vivo are unclear. To address this, we investigated the spread of clinical Extended Spectrum Beta-Lactamase (ESBL)-producing plasmids in the absence of antibiotics in vitro and in the mouse intestine. We hypothesised that plasmid properties would be the primary determinants of plasmid spread and that bacterial strain identity would also contribute. We found clinical Escherichia coli strains natively associated with ESBL-plasmids conjugated to three distinct E. coli strains and one Salmonella enterica serovar Typhimurium strain. Final transconjugant frequencies varied across plasmid, donor, and recipient combinations, with qualitative consistency when comparing transfer in vitro and in vivo in mice. In both environments, transconjugant frequencies for these natural strains and plasmids covaried with the presence/absence of transfer genes on ESBL-plasmids and were affected by plasmid incompatibility. By moving ESBL-plasmids out of their native hosts, we showed that donor and recipient strains also modulated transconjugant frequencies. This suggests that plasmid spread in the complex gut environment of animals and humans can be predicted based on in vitro testing and genetic data.Subject terms: Antibiotics, Microbial ecology, Phylogenomics     

Targeted integration by homologous recombination enables in situ tagging and replacement of genes in the marine microeukaryote Diplonema papillatum

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one, Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination in D. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5′ and 3′ homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of α- and β-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging of D. papillatum genes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.     

Hieracienfunde in den österreichischen Alpen

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Hieracienfunde in den österreichischen Alpen

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Hieracienfunde in den österreichischen Alpen und in der Tatra

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Properties of lipid bilayer membranes made from lipids containing phytanic acid

Besides the preparation of phytanic acid (3,7,11,15-tetramethylhexadecylic acid) according to the Dumas-Stass reaction, the synthesis of four different lipids containing phytanic acid residues is described. Diphytanoyl phophatidylcholine was systhesized beginning from glycerylphosphorylcholine, whereas the other lipids, diphytanoyl phosphatidylethanolamine, diphytanoyl phosphatidylserine and monophytanoyl glyceride were prepared by total synthesis.Some properties of lipid bilayer membranes made from the lipids containing phytanic acid were investigated. The specific capacity of these membranes was measured. Its value of approximately 400 nF cm^?2 was found to be similar to the value of membranes from lipids with unbranched fatty acid residues. Charge pulse experiments were performed using dipicrylamine as a molecular probe of membrane structure. The results were discussed on the basis of a higher viscosity of the membranes from lipids containing phytanic acid residues compared with unbranched fatty acid residues.