Enhancement of gene detection frequencies by combining DNA-based stable-isotope probing with the construction of metagenomic DNA libraries

Summary DNA-based stable-isotope probing (SIP) using ¹³C-labeled growth substrates as bait is a powerful tool for the selective DNA isolation from microorganisms that are actively involved in consuming these substrates. To enhance the detection frequency of target genes in screens for new natural products, we have combined for the first time DNA-based SIP with the construction of metagenomic libraries. To isolate genes encoding coenzyme B₁₂-dependent glycerol dehydratases an enrichment of glycerol-fermenting microorganisms from a sediment sample of the Wadden Sea was performed by using glycerol–¹³C₃ as sole carbon source. Subsequently, the ¹³C-labeled DNA was separated from the naturally abundant ¹²C-DNA by density centrifugation, and used for library generation. Screening of the constructed libraries for the target genes revealed that the gene detection frequencies employing DNA-based SIP for enrichment of genomes harboring dehydratase genes were 2.1- to 3.8-fold higher than those recorded by using a traditional step with unlabeled glycerol for enrichment.     

Regulation of the epithelial Na+ channel by the protein kinase CK2

CK2 is a ubiquitous, pleiotropic, and constitutively active Ser/Thr protein kinase that controls protein expression, cell signaling, and ion channel activity. Phosphorylation sites for CK2 are located in the C terminus of both beta- and gamma-subunits of the epithelial Na(+) channel (ENaC). We examined the role of CK2 on the regulation of both endogenous ENaC in native murine epithelia and in Xenopus oocytes expressing rENaC. In Ussing chamber experiments with mouse airways, colon, and cultured M1-collecting duct cells, amiloride-sensitive Na(+) transport was inhibited dose-dependently by the selective CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB). In oocytes, ENaC currents were also inhibited by TBB and by the structurally unrelated inhibitors heparin and poly(E:Y). Expression of a trimeric channel lacking both CK2 sites (alphabeta(S631A)gamma(T599A)) produced a largely attenuated amiloride-sensitive whole cell conductance and rendered the mutant channel insensitive to CK2. In Xenopus oocytes, CK2 was translocated to the cell membrane upon expression of wt-ENaC but not of alphabeta(S631A)gamma(T599A)-ENaC. Phosphorylation by CK2 is essential for ENaC activation, and to a lesser degree, it also controls membrane expression of alphabetagamma-ENaC. Channels lacking the Nedd4-2 binding motif in beta-ENaC (R561X, Y618A) no longer required the CK2 site for channel activity and siRNA-knockdown of Nedd4-2 eliminated the effects of TBB. This implies a role for CK2 in inhibiting the Nedd4-2 pathway. We propose that the C terminus of beta-ENaC is targeted by this essential, conserved pleiotropic kinase that directs its constitutive activity toward many cellular protein complexes.     

Variation in life-history traits among Urtica dioica populations with different history in parasitism by the holoparasitic plant Cuscuta europaea

Several studies demonstrate that natural enemies (e.g. parasites) have profound negative effects on the life-history traits of their hosts. If the host can compensate for the negative effects of parasitic infection by altering its life history, these modifications may partly form the basis of resistance or tolerance against parasites. Thus, parasites may be of considerable importance in shaping the evolution of life-history traits of their hosts. To examine if previous parasitism is associated with differences in life-history traits of the host, I conducted a common garden experiment with Urtica dioica plants originating from eight populations of which four were unparasitized, and four parasitized by the holoparasitic plant, Cuscuta europaea. A field survey indicated no differences between unparasitized and parasitized populations in, for example, the number of plant species and nutrient levels in the soil. Thus, it seems reasonable to assume that differences in life-history traits between the two population types in the common garden would reflect the effects of previous selection by the parasite. In the common garden, plants from parasitized populations started to flower later and allocated less biomass to asexual reproduction (measured as the production of stolons, i.e. clonal propagation) compared to plants from unparasitized populations. These results thus indicate that selection by the parasite may have favoured later onset of flowering, and may have selected against asexual reproduction.     

The HUPO Brain Proteome project wish list--summary of the 9(th) HUPO BPP Workshop 9-10 January 2008, Barbados

The Human Brain Proteome Project (HUPO BPP) aims at advancing knowledge and the understanding of neurodiseases and aging with the purpose of identifying prognostic and diagnostic biomarkers, as well as to push new diagnostic approaches and medications. The participating groups meet in semi-annual workshops to discuss the progress, as well as the needs, within the field of proteomics. The 9(th) HUPO BPP workshop took place in Barbados from 9-10 January, 2008. Discussing the future HUPO BPP Roadmap, the attendees drafted the so called HUPO BPP wish list containing timelines, suggestions and missions. This wish list will be updated regularly and will serve as a guideline for the next phase.