Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

Elementary steps of the (Na+ + K+)-ATPase mechanism, studied with formycin nucleotides.

1. Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a substrate for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), with properties similar to those of ATP. 2. FTP and formycin diphosphate (FDP) bind to the enzyme with high affinity and, on binding, the nucleotide fluorescence is enhanced 3-4-fold. It is therefore possible, with a stopped-flow fluorimeter, to measure the rates of binding and release of FTP and FDP under conditions in which turnover does not occur. 3. When the enzyme-FTP complex is exposed to conditions permitting turnover (Mg2+, Na+ +/- K+), changes in fluorescence occur which can be explained by supposing that they reflect the interconversion of states with or without bound nucleotides. A rapid fall in fluorescence, that we attribute to the rapid release of FDP from newly phosphorylated enzyme, is followed by a steady state in which low fluorescence suggests that little nucleotide is bound. Eventually, exhaustion of FTP allows rebinding of FDP to the enzyme, which is signalled by a rise in fluorescence. 4. The estimated rate of FDP release from newly formed phosphoenzyme is unaffected by the presence of K+ (0-2 mM) or the concentration of FTP (1-20 micron). 5. Experiments with [gamma-32P]FTP show that about 1 mol of 32P is incorporated per mol of enzyme. The rate of phosphorylation of the enzyme by [gamma-32P]FTP has been measured with a rapid-mixing-and-quenching apparatus. 6. Kinetic data from the fluorescence and phosphorylation experiments show that the behaviour of the enzyme, at least at the low nucleotide concentrations employed, is consistent with the Albers-Post model, and is difficult to reconcile with models in which K+ acts at or before the step in which FDP is released during turnover.     

High-Resolution Physical Map of the X-linked Retinoschisis Interval in Xp22

X-linked retinoschisis (RS) is the leading cause of macular degeneration in young males and has been mapped to Xp22 between DXS418 and DXS999. To facilitate identification of the RS gene, we have constructed a yeast artificial chromosome (YAC) contig across this region comprising 28 YACs and 32 sequence-tagged sites including seven novel end clone markers. To establish the definitive marker order, a PAC contig containing 50 clones was also constructed, and all clones were fingerprinted. The marker order is: Xpter–DXS1317–(AFM205yd12–DXS7175–DXS7992)–60N8T7–DXS1195–DXS7993–DXS7174–60N8-SP6–DXS418–DXS7994–DXS7995–DXS7996–HYAT2–25HA10R–HYAT1–DXS7997–DXS7998–DXS257–434E8R–3542R–DXS6762–DXS7999–DXS6763–434E8L–DXS8000–DXS6760–DXS7176–DXS8001–DXS999–3176R–PHKA2–Xcen. A long-range restriction map was constructed, and the RS region is estimated to be 1300 kb, containing three putative CpG islands. An unstable region was identified between DXS6763 and 434E8L. These data will facilitate positional cloning of RS and other disease genes in Xp22.     

Old trees contribute bio-available nitrogen through canopy bryophytes

Symbiotic cyanobacteria??bryophyte associations on the forest floor are shown to contribute significantly to stand-level nitrogen budgets through the process of biological nitrogen fixation (BNF), but few studies have considered the role of canopy bryophytes. Given the high biomass of epiphytic bryophytes in many tree species of the North American temperate rain forest, we suggest that canopy bryophytes may contribute substantially to stand-level N dynamics. We confirm the presence of cyanobacteria and measure rates of BNF at three heights (0, 15 and 30 m) in Sitka spruce trees across three watershed estuaries of Clayoquot Sound, British Columbia, Canada. This study is the first to report BNF by cyanobacteria associated with epiphytic and forest floor bryophytes in the coastal temperate rain forest of North America. Cyanobacteria density was significantly greater in epiphytic bryophytes compared to mosses on the forest floor, and rates of BNF were highest at 30 m in the canopy. The majority of total stand-level BNF (0.76 kg N · ha^-1 · yr^-1) occurs in the canopy, rather than on the forest floor (0.26 kg N · ha^-1 · yr^-1). We suggest that BNF by cyanobacterial-bryophyte associations in the canopy of coastal temperate rain forests is a unique source of ecosystem N, which is dependent on large, old trees with high epiphytic bryophyte biomass.     

Detecting RNA viruses in living mammalian cells by fluorescence microscopy

Traditional methods that rely on viral isolation and culture techniques continue to be the gold standards used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells.     

Chemical and biological approaches synergize to ameliorate protein-folding diseases

Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.