The 3′ Untranslated Region of the Rabies Virus Glycoprotein mRNA Specifically Interacts with Cellular PCBP2 Protein and Promotes Transcript Stability

Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA stability. Reporter assays performed both in electroporated cells and in a cell-free RNA decay system indicate that the conserved portion of the 3′ UTR of the rabies virus glycoprotein mRNA contains an RNA stability element. PCBP2 specifically interacts with reporter transcripts containing this 72 base 3′ UTR sequence. Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts. Therefore, we conclude that PCBP2 specifically and selectively interacts with the rabies virus glycoprotein mRNA and that this interaction may contribute to the post-transcriptional regulation of glycoprotein expression

Monitoring of microbial hydrocarbon remediation in the soil

Bioremediation of hydrocarbon pollutants is advantageous owing to the cost-effectiveness of the technology and the ubiquity of hydrocarbon-degrading microorganisms in the soil. Soil microbial diversity is affected by hydrocarbon perturbation, thus selective enrichment of hydrocarbon utilizers occurs. Hydrocarbons interact with the soil matrix and soil microorganisms determining the fate of the contaminants relative to their chemical nature and microbial degradative capabilities, respectively. Provided the polluted soil has requisite values for environmental factors that influence microbial activities and there are no inhibitors of microbial metabolism, there is a good chance that there will be a viable and active population of hydrocarbon-utilizing microorganisms in the soil. Microbial methods for monitoring bioremediation of hydrocarbons include chemical, biochemical and microbiological molecular indices that measure rates of microbial activities to show that in the end the target goal of pollutant reduction to a safe and permissible level has been achieved. Enumeration and characterization of hydrocarbon degraders, use of micro titer plate-based most probable number technique, community level physiological profiling, phospholipid fatty acid analysis, 16S rRNA- and other nucleic acid-based molecular fingerprinting techniques, metagenomics, microarray analysis, respirometry and gas chromatography are some of the methods employed in bio-monitoring of hydrocarbon remediation as presented in this review

Characterization of methanogenic and methanotrophic assemblages in landfill samples.

A greater understanding of the tightly linked trophic groups of anaerobic and aerobic bacteria residing in municipal solid waste landfills will increase our ability to control methane emissions and pollutant fate in these environments. To this end, we characterized the composition of methanogenic and methanotrophic bacteria in samples taken from two regions of a municipal solid waste landfill that varied in age. A method combining polymerase chain reaction amplification, restriction fragment length polymorphism analysis and phylogenetic analysis was used for this purpose. 16S rDNA sequence analysis revealed a rich assemblage of methanogens in both samples, including acetoclasts, H2/CO2-users and formate-users in the newer samples and H2/CO2-users and formate-users in the older samples, with closely related genera including Methanoculleus, Methanofollis, Methanosaeta and Methanosarcina. Fewer phylotypes of type 1 methanotrophs were observed relative to type 2 methanotrophs. Most type 1 sequences clustered within a clade related to Methylobacter, whereas type 2 sequences were broadly distributed among clades associated with Methylocystis and Methylosinus species. This genetic characterization tool promises rapid screening of landfill samples for genotypes and, therefore, degradation potentials

Simultaneous Recovery of RNA and DNA from Soils and Sediments

Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316–322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0°C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 μg g of soil(−1) dry weight), whereas the DNA yield ranged from 23 to 435 μg g(−1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification

Nonexhaustive cyclodextrin-based extraction technique for the evaluation of PAH bioavailability.

Traditionally, soil extraction techniques have been concerned with the determination of ``total'' organic contaminant concentrations, following an ``exhaustive'' extraction. However, in light of the increasing body of knowledge relating to organic contaminant availability and aging, such methods have little relevance to the amount of contaminant that may pose an ecological risk i.e., the ``bioavailable'' portion. Less exhaustive techniques have therefore been the subject of more recent approaches in the hope that they may access the ``labile'' or bioavailable pool. The use of an aqueous-based extraction technique utilizing hydroxypropyl-beta-cyclodextrin (HPCD) is presented here for the extraction of PAHs from soil. The optimization of the method is described in terms of HPCD concentration, extraction time, and solution buffering. The procedure is then tested and validated for a range of C-14-labeled PAHs (phenanthrene, pyrene, and benzo[a]pyrene) added at a range of concentrations to a range of soil types. The amounts of soil-associated phenanthrene mineralized by catabolically active microorganisms were correlated with total residual phenanthrene concentrations (r(2) = 0.889; slope of best fit line = 0.763; intercept = -5.662; n = 24), dichloromethane (DCM)-extractable phenanthrene concentrations (r2 = 0.986; slope of best fit line = 0.648; intercept = 0.340; n = 24), butan-1-ol (BuOH)-extractable phenanthrene concentrations (r(2) = 0.957; slope of best fit line = 0.614; intercept = 0.544; n = 24), and HPCD-extractable phenanthrene concentrations (r(2) = 0.964; slope of best fit line = 0.997; intercept = 0.162; n = 24). Th us, in this study, the microbially bioavailable concentrations of soil-associated phenanthrene were best predicted using the optimized HPCD extraction technique. In contrast, the DCM Soxhlet extraction and the BuOH shake extraction both overestimated phenanthrene bioavailability by, on average, >60%.