Evolutionary Evidence for Alternative Structure in RNA Sequence Co-variation

Sequence conservation and co-variation of base pairs are hallmarks of structured RNAs. For certain RNAs (e.g. riboswitches), a single sequence must adopt at least two alternative secondary structures to effectively regulate the message. If alternative secondary structures are important to the function of an RNA, we expect to observe evolutionary co-variation supporting multiple conformations. We set out to characterize the evolutionary co-variation supporting alternative conformations in riboswitches to determine the extent to which alternative secondary structures are conserved. We found strong co-variation support for the terminator, P1, and anti-terminator stems in the purine riboswitch by extending alignments to include terminator sequences. When we performed Boltzmann suboptimal sampling on purine riboswitch sequences with terminators we found that these sequences appear to have evolved to favor specific alternative conformations. We extended our analysis of co-variation to classic alignments of group I/II introns, tRNA, and other classes of riboswitches. In a majority of these RNAs, we found evolutionary evidence for alternative conformations that are compatible with the Boltzmann suboptimal ensemble. Our analyses suggest that alternative conformations are selected for and thus likely play functional roles in even the most structured of RNAs.     

Arg343 in human surfactant protein D governs discrimination between glucose and N-acetylglucosamine ligands

Surfactant protein D (SP-D), one of the members of the collectin family of C-type lectins, is an important component of pulmonary innate immunity. SP-D binds carbohydrates in a calcium-dependent manner, but the mechanisms governing its ligand recognition specificity are not well understood. SP-D binds glucose (Glc) stronger than N-acetylglucosamine (GlcNAc). Structural superimposition of hSP-D with mannose- binding protein C (MBP-C) complexed with GlcNAc reveals steric clashes between the ligand and the side chain of Arg343 in hSP-D. To test whether Arg343 contributes to Glc > GlcNAc recognition specificity, we constructed a computational model of Arg343-->Val (R343V) mutant hSP-D based on homology with MBP-C. Automated docking of alpha-Me-Glc and alpha-Me-GlcNAc into wild-type hSP-D and the R343V mutant of hSP-D suggests that Arg343 is critical in determining ligand-binding specificity by sterically prohibiting one binding orientation. To empirically test the docking predictions, an R343V mutant recombinant hSP-D was constructed. Inhibition analysis shows that the R343V mutant binds both Glc and GlcNAc with higher affinity than the wild-type protein and that the R343V mutant binds Glc and GlcNAc equally well. These data demonstrate that Arg343 is critical for hSP-D recognition specificity and plays a key role in defining ligand specificity differences between MBP and SP-D. Additionally, our results suggest that the number of binding orientations contributes to monosaccharide binding affinity.     

Species Composition of Subalpine Grassland is Sensitive to Nitrogen Deposition, but Not to Ozone, After Seven Years of Treatment

Reactive nitrogen (N) and ozone (O₃) are the most widespread atmospheric pollutants with significant implications for conservation of semi-natural vegetation; their combined effects have, however, not been tested in long-term field experiments. To investigate these effects on the species composition of a subalpine Geo-Montani-Nardetum pasture, 180 turf monoliths were exposed for seven years to five N loads (0, +5, +10, +25, +50 kg N ha^?1 y^?1) in combination with three O₃ levels (ambient, 1.2 or 1.6× ambient concentration) in a free-air fumigation experiment at 2000 m a.s.l. in the Central Alps. Aboveground biomass of grasses, forbs, sedges, and legumes, as well as individual species abundance was recorded annually. N addition caused strong changes in community composition and slightly reduced Shannon diversity: Sedges (Carex sempervirens and Carex ornithopoda) tripled their fractional biomass at the expense of legumes (Trifolium alpinum), grasses (Agrostis capillaris, Briza media, Festuca spp.), and forbs, the latter of which responded inconsistently. Compositional changes were significant with +5 kg ha^?1 y^?1; at all levels of N, however, changes ceased after 5 years. Elevated O₃ and the combined O₃ × N exposure had no effect on functional group productivity. Overall the results reveal high N sensitivity of the subalpine grassland, but low sensitivity to O_3, singly or in combination with N. Thus, in the longer term any input of N above the current ambient deposition may cause a shift in the plant community composition of these ecosystems which are considered hotspots for biodiversity.     

The CD95 receptor: apoptosis revisited

CD95 is the quintessential death receptor and, when it is bound by ligand, cells undergo apoptosis. Recent evidence suggests, however, that CD95 mediates not only apoptosis but also diverse nonapoptotic functions depending on the tissue and the conditions.     

Crystal structure of osmoporin OmpC from E. coli at 2.0 A

Porins form transmembrane pores in the outer membrane of Gram-negative bacteria with matrix porin OmpF and osmoporin OmpC from Escherichia coli being differentially expressed depending on environmental conditions. The three-dimensional structure of OmpC has been determined to 2.0 A resolution by X-ray crystallography. As expected from the high sequence similarity, OmpC adopts the OmpF-like 16-stranded hollow beta-barrel fold with three beta-barrels associated to form a tight trimer. Unlike in OmpF, the extracellular loops form a continuous wall at the perimeter of the vestibule common to the three pores, due to a 14-residues insertion in loop L4. The pore constriction and the periplasmic outlet are very similar to OmpF with 74% of the pore lining residues being conserved. Overall, only few ionizable residues are exchanged at the pore lining. The OmpC structure suggests that not pore size, but electrostatic pore potential and particular atomic details of the pore linings are the critical parameters that physiologically distinguish OmpC from OmpF.     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

Phylogenetic analysis of Pinguicula (Lentibulariaceae): chloroplast DNA sequences and morphology support several geographically distinct radiations

The genus Pinguicula is one of the three genera of the carnivorous Lentibulariaceae, comprising approximately 80 species. Phylogeny inference using nucleotide sequences of the chloroplast gene matK and the trnK group II intron, as well as a set of 32 morphological characters revealed five well-supported, major lineages within the genus. These lineages largely reflect radiations in clearly defined geographic regions, whereas most previously recognized sections of the genus are shown to be para- or polyphyletic. A species-rich Mexican-Central American-Caribbean clade has the Eurasian P. alpina and an East Asian clade as successive sisters. All three are characterized by a production of flower buds on winter-resting plants, a specific corolla hair structure and a very large corolla lower central lobe. Another diverse clade is composed of species with primarily European distribution including the widespread type species P. vulgaris. For this clade, vegetative reproduction during dormancy is synapomorphic. Species native to SE North America and the South American Andes and a group of Mediterranean and NE Atlantic coast species together appear in a fifth well-supported clade, that is characterized by a tropical-type growth habit. It is the only clade that has reached temperate zones of the southern hemisphere.