Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules

Shrub Invasion Decreases Diversity and Alters Community Stability in Northern Chihuahuan Desert Plant Communities

BACKGROUND:Global climate change is rapidly altering species range distributions and interactions within communities. As ranges expand, invading species change interactions in communities which may reduce stability, a mechanism known to affect biodiversity. In aridland ecosystems worldwide, the range of native shrubs is expanding as they invade and replace native grassland vegetation with significant consequences for biodiversity and ecosystem functioning. METHODOLOGY:We used two long-term data sets to determine the effects of shrub encroachment by Larrea tridentata on subdominant community composition and stability in formerly native perennial grassland dominated by Bouteloua eriopoda in New Mexico, USA. PRINCIPAL FINDINGS:Our results indicated that Larrea invasion decreased species richness during the last 100 years. We also found that over shorter temporal scales species-poor subdominant communities in areas invaded by Larrea were less stable (more variable in time) compared to species rich communities in grass-dominated vegetation. Compositional stability increased as cover of Bouteloua increased and decreased as cover of Larrea increased. SIGNIFICANCE:Changes in community stability due to altered interspecific interactions may be one mechanism by which biodiversity declines in grasslands following shrub invasion. As global warming increases, shrub encroachment into native grasslands worldwide will continue to alter species interactions and community stability both of which may lead to a decline in biodiversity

Cathepsin-L can resist lysis by human serum in Trypanosoma brucei brucei.

Closely related African trypanosomes cause lethal diseases but display distinct host ranges. Specifically, Trypanosoma brucei brucei causes nagana in livestock but fails to infect humans, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause sleeping sickness in humans. T. b. brucei fails to infect humans because it is sensitive to innate immune complexes found in normal human serum known as trypanolytic factor (TLF) 1 and 2; the lytic component is apolipoprotein-L1 in both TLFs. TLF resistance mechanisms of T. b. gambiense and T. b. rhodesiense are now known to arise through either gain or loss-of-function, but our understanding of factors that render T. b. brucei susceptible to lysis by human serum remains incomplete. We conducted a genome-scale RNA interference (RNAi) library screen for reduced sensitivity to human serum. Among only four high-confidence 'hits' were all three genes previously shown to sensitize T. b. brucei to human serum, the haptoglobin-haemoglobin receptor (HpHbR), inhibitor of cysteine peptidase (ICP) and the lysosomal protein, p67, thereby demonstrating the pivotal roles these factors play. The fourth gene identified encodes a predicted protein with eleven trans-membrane domains. Using chemical and genetic approaches, we show that ICP sensitizes T. b. brucei to human serum by modulating the essential cathepsin, CATL, a lysosomal cysteine peptidase. A second cathepsin, CATB, likely to be dispensable for growth in in vitro culture, has little or no impact on human-serum sensitivity. Our findings reveal major and novel determinants of human-serum sensitivity in T. b. brucei. They also shed light on the lysosomal protein-protein interactions that render T. b. brucei exquisitely sensitive to lytic factors in human serum, and indicate that CATL, an important potential drug target, has the capacity to resist these factors

Rewetting CO2 pulses in Australian agricultural soils and the influence of soil properties

An incubation study determined the effect of one dry–rewetting (DRW) event on the turnover of carbon (C), phosphorus (P) and nitrogen (N). Thirty-two soils were collected from different climatic regions of southern Australia, varying in soil type, land use and agronomic management history. We hypothesised that respiration and nutrient pulses are related to soil physio-chemical properties. Respiration (CO2 release) was measured intensively for 90 h after rewetting. C mineralisation (C min) model fitting was used to describe the amount of mineralisable C (Co90 h) and the proportional mineralisation rate (k). Compared to constantly moist soils, 13 soils showed increases in both Co90 h and k, indicating that DRW increased the amount of mineralisable C and the rate at which C was mineralised over the 90-h period. In 17 soils, k was increased but not Co90 h, showing an increase in C mineralisation rate but no change in the amount of mineralisable C. Two soils showed a reduction in k with no change in Co90 h, possibly due to low C contents and small microbial biomass. Only one soil exhibited no change in either Co90 h or k. Multiple linear regression analysis indicated that the magnitude of the increase in mineralisable C in response to the DRW event (∆Co90 h = Co90 h DRW − Co90 h moist) was primarily explained by clay content (39%); however, inclusion of nine soil physio-chemical properties explained more of the variation in ∆Co90 h than any of the properties alone. Five of the nine physio-chemical variables present in the multiple-regression model were related to C content or composition. Pulses in available N and P were not related to ∆Co90 h.Clayton R. Butterly, Petra Marschner, Ann M. McNeill and Jeff A. Baldoc

Drying and wetting in saline and saline-sodic soils - effects on microbial activity, biomass and dissolved organic carbon

Aims: There are few studies on the interactive effect of salinity and sodicity in soils exposed to drying and wetting cycles. We conducted a study to assess the impact of multiple drying and wetting on microbial respiration, dissolved organic carbon and microbial biomass in saline and saline-sodic soils. Methods: Different levels of salinity (EC 1:5 1.0 or 2.5) and sodicity (SAR < 3 or 20) were induced by adding NaCl and CaCl 2 to a non-saline/non-sodic soil. Finely ground wheat straw residue was added at 20 g kg -1 as substrate to stimulate microbial activity. The constant moist (CM) treatment was kept at optimum moisture content for the length of the experiment. The drying and rewetting (DW) treatments consisted of 1 to 3 DW cycles; each DW cycle consisted of 1 week drying after which they were rewet to optimum moisture and then maintained moist for 1 week. Results: Drying reduced respiration more strongly at EC2.5 than with EC1.0. Rewetting of dry soils produced a flush in respiration which was greatest in the soils without salt addition and smallest at high salinity (EC2.5) suggesting better substrate utilisation by microbes in soils without added salts. After three DW events, cumulative respiration was significantly increased by DW compared to CM, being 24% higher at EC1.0 and 16% higher at EC2.5 indicating that high respiration rates after rewetting may compensate for the low respiration rates during the dry phase. The respiration rate per unit MBC was lower at EC2.5 than at EC1.0. Further, the size of the flush in respiration upon rewetting decreased with each ensuing DW cycle being 50-70% lower in the third DW cycle than the first. Conclusions: Both salinity and sodicity alter the effect of drying and rewetting on soil carbon dynamics compared to non-saline soils. © 2011 Springer Science+Business Media B.V.Manpreet S. Mavi, Petra Marschne