Heterogeneity of a fluorescent tegument component in single pseudorabies virus virions and enveloped axonal assemblies

The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.     

The location of eggs retained in the oviducts of mares

The oviducts of 24 mares were examined to determine the site of retention of unfertilized eggs. The ampullary-isthmic junction regions of 42 of the 48 oviducts were serially sectioned and examined histologically. The remaining parts of the oviducts were flushed and the flushings searched microscopically. Of 45 eggs located, 40 were in the sectioned segments of 24 oviducts and only 5 were in the flushings. All but one of the sectioned segments contained prominent masses of material obstructing the lumen, but these were apparently not the direct cause of egg retention since eggs were found on both sides of them.     

Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.     

Analysis of hapten-specific T suppressor factors: genetic restriction of TsF1 activity analyzed with synthetic hybrid suppressor molecules

We have analyzed the first-order suppressor factor secreted by an azobenzenearsonate (ABA)-specific T suppressor cell (Ts) hybridoma. Treatment of the factor with 5 mM dithiothreitol (DTT) yields two fragments with distinct phenotypes and functional capabilities. One fragment is bound by a monoclonal anti-I-J antibody, the other is not. Further, although neither molecular fragment by itself is sufficient to suppress an ABA response, a mixture of the two reconstitutes the suppressive activity. The I-J- portion of the first-order suppressor factor (TsF1) presumably guides the antigen specificity; activity of the ABA-specific Ts I-J- TsF1 factor can be reconstituted with an I-J+ subunit of a TsF molecule of either sheep red blood cell (SRBC) or ABA specificity. The genetic restriction for Igh-linked determinants of the ABA/SRBC hybrid TsF molecules is influenced by the I-J+ portion, regardless of the original antigen specificity of that molecule. The data support a two-subunit TsF model. Polyclonal ABA-specific TsF1 molecules appear to resemble the monoclonal factor in structure.     

Invasion impacts on functions and services of aquatic ecosystems

Hydrobiologia - Non-native species can simultaneously affect ecological structures, functions, and services of the invaded ecosystem. In this paper, we report that the study of non-native...     

Theoretical ab Initio Study of the Effects of Methylation on Structure and Stability of G:C Watson-Crick Base Pair

Abstract

Methylation of DNA occurs most readily at N(3), N(7), and O(6) of purine bases and N(3) and O(2) of pyrimidines. Methylated bases are continuously formed through endogenous and exogenous mechanisms. The results of a theoretical ab initio study on the methylation of G:C base pair components are reported. The geometries of the local minima were optimized without symmetry restrictions by the gradient procedure at DFT level of theory and were verified by energy second derivative calculations. The standard 6–31G(d) basis set was used. The single-point calculations have been performed at the MP2/6–31G(d,p), MP2/6–31₊₊G(d,p), and MP2/6–311₊₊G(2d,2p) levels of theory. The geometrical parameters, relative stability and counterpoise corrected interaction energies are reported. Also, using a variation-perturbation energy decomposition scheme we have found the vital contributions to the total interaction energy.     

Barite encrustation of benthic sulfur‐oxidizing bacteria at a marine cold seep

Crusts and chimneys composed of authigenic barite are found at methane seeps and hydrothermal vents that expel fluids rich in barium. Microbial processes have not previously been associated with barite precipitation in marine cold seep settings. Here, we report on the precipitation of barite on filaments of sulfide‐oxidizing bacteria at a brine seep in the Gulf of Mexico. Barite‐mineralized bacterial filaments in the interiors of authigenic barite crusts resemble filamentous sulfide‐oxidizing bacteria of the genus Beggiatoa. Clone library and iTag amplicon sequencing of the 16S rRNA gene show that the barite crusts that host these filaments also preserve DNA of Candidatus Maribeggiatoa, as well as sulfate‐reducing bacteria. Isotopic analyses show that the sulfur and oxygen isotope compositions of barite have lower δ³⁴S and δ¹⁸O values than many other marine barite crusts, which is consistent with barite precipitation in an environment in which sulfide oxidation was occurring. Laboratory experiments employing isolates of sulfide‐oxidizing bacteria from Gulf of Mexico seep sediments showed that under low sulfate conditions, such as those encountered in brine fluids, sulfate generated by sulfide‐oxidizing bacteria fosters rapid barite precipitation localized on cell biomass, leading to the encrustation of bacteria in a manner reminiscent of our observations of barite‐mineralized Beggiatoa in the Gulf of Mexico. The precipitation of barite directly on filaments of sulfide‐oxidizing bacteria, and not on other benthic substrates, suggests that sulfide oxidation plays a role in barite formation at certain marine brine seeps where sulfide is oxidized to sulfate in contact with barium‐rich fluids, either prior to, or during, the mixing of those fluids with sulfate‐containing seawater in the vicinity of the sediment/water interface. As with many other geochemical interfaces that foster mineral precipitation, both biological and abiological processes likely contribute to the precipitation of barite at marine brine seeps such as the one studied here.