Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb³⁺ ions and the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb³⁺ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem–loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.     

Identification of Differentially Expressed Genes Associated with Osteochondrosis in Standardbred Horses Using RNA Arbitrarily Primed PCR

The aim of this study was to investigate genes for differential expression in cartilage of foals predisposed to osteochondrosis (OC). Tissue was sampled from the cranial part of the distal intermediate ridge of the tibia in the tarso-crural joint. Foals were considered predisposed to OC when parents had OC at the distal intermediate ridge of the tibia. RNA was isolated and subjected to arbitrarily primed PCR (RAP-PCR) followed by fingerprinting to screen for differentially expressed genes. By verification of results from the RAP-PCR fingerprint screening using real-time RT-PCR, we identified two genes not previously correlated with OC as differentially expressed. The two genes, which were identical to TLK2 and an equine EST, are good targets for future research on OC.     

A ToF-SIMS study of the lateral organization of lipids and proteins in pulmonary surfactant systems

Pulmonary surfactant is a complex lipid-protein mixture whose main function is to reduce the surface tension at the air-liquid interface of alveoli to minimize the work of breathing. The exact mechanism by which surfactant monolayers and multilayers are formed and how they lower surface tension to very low values during lateral compression remains uncertain. We used time-of-flight secondary ion mass spectrometry to study the lateral organization of lipids and peptide in surfactant preparations ranging in complexity. We show that we can successfully determine the location of phospholipids, cholesterol and a peptide in surfactant Langmuir-Blodgett films and we can determine the effect of cholesterol and peptide addition. A thorough understanding of the lateral organization of PS interfacial films will aid in our understanding of the role of each component as well as different lipid-lipid and lipid-protein interactions. This may further our understanding of pulmonary surfactant function.     

The role of El Niño–Southern Oscillation in the dynamics of a savanna large herbivore population

Our understanding of large‐scale climatic phenomena and dynamics of large herbivore populations comes principally from research in northern regions with temperate, seasonal climate and animal communities with relatively low species diversity. To assess the generality of that perspective, we investigated effects of El Niño–Southern Oscillation (ENSO) on population dynamics of African buffalo Syncerus caffer inhabiting a semi‐arid savanna with variable rainfall. We used linear and nonlinear‐threshold models to investigate relationships between population parameters and explanatory variables affecting forage conditions (seasonal rainfall, Southern Oscillation Index [SOI]). El Niño‐related droughts in 1982–1983 and 1991–1992 were associated with strongly negative population change, a pattern expected to coincide with a decrease in normally high and constant adult survival. Consistent with that nonlinear pattern, we detected threshold relationships between wet‐season rainfall and population change. Juvenile recruitment was described best by linear relationships with dry‐season. Because ENSO operates primarily through wet‐season rainfall, whereas population dynamics were also related to dry‐season rainfall, SOI did not have the predictive ability of individual weather components.     

A unique role of the cholera toxin A1-DD adjuvant for long-term plasma and memory B cell development

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ～36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.     

Viral RNAi suppressor reversibly binds siRNA to outcompete Dicer and RISC via multiple turnover

RNA interference is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response to viruses and retrotransposons. During viral infection, the RNase-III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs) 21-24 nucleotides in length and helps load them into the RNA-induced silencing complex (RISC) to guide the cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressors (RSS) that tightly, and presumably quantitatively, bind siRNAs to thwart RNA-interference-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus, as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding [(1.69 ± 0.07) × 10⁸ M^− 1 s^− 1] and marked dissociation (k_off = 0.062 ± 0.002 s^− 1). We also observe that p19 efficiently competes with recombinant Dicer and inhibits the formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy.     

Management strategy evaluation: a powerful tool for conservation?

The poor management of natural resources has led in many cases to the decline and extirpation of populations. Recent advances in fisheries science could revolutionize management of harvested stocks by evaluating management scenarios in a virtual world by including stakeholders and by assessing its robustness to uncertainty. These advances have been synthesized into a framework, management strategy evaluation (MSE), which has hitherto not been used in terrestrial conservation. We review the potential of MSE to transform terrestrial conservation, emphasizing that the behavior of individual harvesters must be included because harvester compliance with management rules has been a major challenge in conservation. Incorporating resource user decision-making required to make MSEs relevant to terrestrial conservation will also advance fisheries science.