Atomic force microscope measurements of long-range forces near lipid-coated surfaces in electrolytes.

The interaction of DMPC (L-alpha-dimyristoyl-1,2-diterradecanoyl-sn-glycero-3-phosphoch oli ne, C36H72NO8P) lipid-coated Si3N4 surfaces immersed in an electrolyte was investigated with an atomic force microscope. A long-range interaction was observed, even when the Si3N4 surfaces were covered with nominally neutral lipid layers. The interaction was attributed to Coulomb interactions of charges located at the lipid surface. The experimental force curves were compared with solutions for the linearized as well as with exact solutions of the Poisson-Boltzmann equation. The comparison suggested that in 0.5 mM KCl electrolyte the DMPC lipids carried about one unit of charge per 100 lipid molecules. The presence of this surface charge made it impossible to observe an effective charge density recently predicted for dipole layers near a dielectric when immersed in an electrolyte. A discrepancy between the theoretical results and the data at short separations was interpreted in terms of a decrease in the surface charge with separation distance.     

N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express.

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.     

Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3′ terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3′ terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3′ cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3′ termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3′ terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3′ vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.     

Phospholipases. I. Effect ofn-alkanols on the rate of enzymatic hydrolysis of egg phosphatidylcholine

Summary The rate of hydrolysis of unsonicated liposomes of egg lecithin by phospholipase A (from bee venom and Russell viper venom) and phospholipase C (fromBacillus cereus andClostridium welchii) is markedly dependent on the nature and concentration of a variety of added alcohols. Typical plots of rate against alcohol concentration are bell-shaped. The maximum rate and the alcohol concentration at which it is achieved are alcohol-specific. In a homologous series ofn-alkanols, the maximal rates increase and the optimal concentrations decrease as the chain length is increased from C₄ to C₈. For longer alcohols (C₉ to C₁₂), progressively higher concentrations are required to elicit maximal activation. The optimal activating concentrationsC for C₄ to C₈ n-alkanols obey the relationshipp C=a logP _octanol+constant [cf. Hansch & Dunn,J. Pharm. Sci. 61:1 (1972)], suggesting that the alcohol-activating effect is a consequence of their incorporation into the liposomes with resultant modification of liposomal structure.     

Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.     

Evidence for a substrate-binding subsite in ribonuclease T1. Crystal structure of the complex with two guanosines, and model building of the complex with the substrate guanylyl-3',5'-guanosine

The enzyme ribonuclease T1 cleaves single-stranded RNA at the 3'-side of guanosine. The structure of the complex with two guanosines has been analyzed at 1.8-A resolution and refined to a crystallographic R value of 14.0%. One guanosine occupies the guanosine recognition site as observed in previously analyzed complexes of ribonuclease T1 with guanosine phosphates. The other is bound to a base-unspecific subsite marking the binding locus of the nucleoside 3'-proximal to guanosine in a cleavable RNA chain. The positions of the guanosine bound to the recognition site and of the guanine base at the subsite were used to guide model building of the substrate guanylyl-3',5'-guanosine bound to the active site of ribonuclease T1. After energy minimization and a 7-ps stochastic dynamics simulation, a plausible model of the enzyme-substrate complex was obtained which may serve as a reference point in consideration of the mechanisms of RNA hydrolysis by ribonuclease T1.