Functional domains of the ubiquitous chromatin protein DEK

DEK was originally described as a proto-oncogene protein and is now known to be a major component of metazoan chromatin. DEK is able to modify the structure of DNA by introducing supercoils. In order to find interaction partners and functional domains of DEK, we performed yeast two-hybrid screens and mutational analyses. Two-hybrid screening yielded C-terminal fragments of DEK, suggesting that DEK is able to multimerize. We could localize the domain to amino acids 270 to 350 and show that multimerization is dependent on phosphorylation by CK2 kinase in vitro. We also found two DNA binding domains of DEK, one on a fragment including amino acids 87 to 187 and containing the SAF-box DNA binding motif, which is located between amino acids 149 and 187. This region is sufficient to introduce supercoils into DNA. The second DNA binding domain is located between amino acids 270 and 350 and thus overlaps the multimerization domain. We show that the two DNA-interacting domains differ in their binding properties and in their abilities to respond to CK2 phosphorylation.     

Sexual harassment in a live-bearing fish (<Emphasis Type="Italic">Poecilia mexicana</Emphasis>): influence of population-specific male mating behaviour

We investigated male sexual behaviour and the cost of sexual harassment, as measured by the reduction of female feeding time in the presence of a male, in a cave-dwelling population of Poecilia mexicana, in which sexual harassment does not occur naturally. We asked whether the lack of sexual harassment in this population is due to low sexual activity of the males, or low feeding motivation of the females. We experimentally increased the sexual activity of males or the females feeding motivation, or we used a combination of both treatments. Female feeding time was not lower in the presence of a male than in the presence of a female after sexual deprivation of the males or food deprivation of the females. Only in the combined experiment was female feeding time lower in the presence of a large male than in the presence of a small male, indicating a weak effect of sexual harassment by large males. Virgin females did not suffer a cost of sexual harassment, indicating that sexual experience does not cause the lack of sexual harassment in cave mollies. Males from a surface population, where sexual harassment occurs, significantly reduced the feeding time of cave-dwelling females even though these males exhibited surprisingly little sexual behaviour. The sexual activity of cave mollies did not correlate with male body size in any experiment, indicating that even after sexual deprivation, small cave molly males do not switch to the alternative mating behaviour known in surface-dwelling P. mexicana, where sexual activity is correlated negatively with male body size.     

Structure–function relationships of four compression wood types: micromechanical properties at the tissue and fibre level

The mechanisms behind compressive stress generation in gymnosperms are not yet fully understood. Investigating the structure–function relationships at the tissue and cell level, however, can provide new insights. Severe compression wood of all species lacks a S3 layer, has a high microfibril angle in the S2 layer and a high lignin content. Additionally, special features like helical cavities or spiral thickenings appear, which are not well understood in terms of their mechanical relevance, but need to be examined with regard to evolutionary trends in compression wood development. Thin compression wood foils and isolated tracheids of four gymnosperm species [Ginkgo biloba L., Taxus baccata L., Juniperus virginiana L., Picea abies (L.) Karst.] were investigated. The tracheids were isolated mechanically by peeling them out of the solid wood using fine tweezers. In contrast to chemical macerations, the cell wall components remained in their original condition. Tensile properties of tissue foils and tracheids were measured in a microtensile apparatus under wet conditions. Our results clearly show an evolutionary trend to a much more flexible compression wood. An interpretation with respect to compressive stress generation is discussed.     

The DEK protein--an abundant and ubiquitous constituent of mammalian chromatin

The protein DEK is an abundant and ubiquitous chromatin protein in multicellular organisms (not in yeast). It is expressed in more than a million copies/nucleus of rapidly proliferating mammalian cells. DEK has two DNA binding modules of which one includes a SAP box, a sequence motif that DEK shares with a number of other chromatin proteins. DEK has no apparent affinity to specific DNA sequences, but preferentially binds to superhelical and cruciform DNA, and induces positive supercoils into closed circular DNA. The available evidence strongly suggests that DEK could function as an architectural protein in chromatin comparable to the better known classic architectural chromatin proteins, the high-mobility group or HMG proteins.     

Adaptive Growth of Gymnosperm Branches-Ultrastructural and Micromechanical Examinations

The top, the lateral and the underside of basal branch segments of two gymnosperm species, spruce (Picea abies [L.] Karst.) and yew (Taxus baccata L.), were studied with respect to possible adaptation in structural and mechanical properties. Microtensile tests were performed on thin wet foils, which were removed from the periphery of the branches. Structural parameters such as density and the microfibril angle in the S2-layer were examined to investigate the structure-function relationships of the branch wood. The top, the lateral and the underside of both branches showed significant differences in their structural and mechanical properties. However, no significant variations were observed as a function of age and size development. The findings were discussed in view of adaptive growth strategies of trees, including biomechanical constraints of a horizontally growing branch.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.     

Structural basis of dynamic glycine receptor clustering by gephyrin

Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.     

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.