Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.     

Characterization of promoter function and cell-type-specific expression from viral vectors in the nervous system

Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.     

Phytoremediation of Mercury- and Methylmercury-Polluted Soils Using Genetically Engineered Plants

Inorganic mercury in contaminated soils and sediments is relatively immobile, though biological and chemical processes can transform it to more toxic and bioavailable methylmercury. Methylmercury is neurotoxic to vertebrates and is biomagnified in animal tissues as it is passed from prey to predator. Traditional remediation strategies for mercury contaminated soils are expensive and site-destructive. As an alternative we propose the use of transgenic aquatic, salt marsh, and upland plants to remove available inorganic mercury and methylmercury from contaminated soils and sediments. Plants engineered with a modified bacterial mercuric reductase gene, merA, are capable of converting Hg(II) taken up by roots to the much less toxic Hg(0), which is volatilized from the plant. Plants engineered to express the bacterial organo-mercurial lyase gene, merB, are capable of converting methylmercury taken up by plant roots into sulfhydryl-bound Hg(II). Plants expressing both genes are capable of converting ionic mercury and methylmercury to volatile Hg(0) which is released into an enormous global atmospheric Hg(0) pool. To assess the phytoremediation capability of plants containing the merA gene, a variety of assays were carried out with the model plants Arabidopsis thaliana, and tobacco (Nicotiana tabacum).     

The physiology and ecological implications of efficient growth

The natural habitats of microbes are typically spatially structured with limited resources, so opportunities for unconstrained, balanced growth are rare. In these habitats, selection should favor microbes that are able to use resources most efficiently, that is, microbes that produce the most progeny per unit of resource consumed. On the basis of this assertion, we propose that selection for efficiency is a primary driver of the composition of microbial communities. In this article, we review how the quality and quantity of resources influence the efficiency of heterotrophic growth. A conceptual model proposing innate differences in growth efficiency between oligotrophic and copiotrophic microbes is also provided. We conclude that elucidation of the mechanisms underlying efficient growth will enhance our understanding of the selective pressures shaping microbes and will improve our capacity to manage microbial communities effectively.     

Hiding in the Shadows: Philip Morris and the Use of Third Parties to Oppose Ingredient Disclosure Regulations

Background

In 1996 Massachusetts proposed regulations that would require tobacco companies to disclose information about the ingredients in their products on a by-brand basis. This paper examines the strategies employed by Philip Morris to stop these regulations from being implemented.

Methods and Finding

We used previously secret tobacco industry documents and published literature to examine the activities of the tobacco companies after the regulations were proposed. Philip Morris hired a public relations firm to establish a coalition that was instructed to oppose the regulations by linking them to other industrial sectors (the slippery slope) and stating they would damage the state''s economy. Philip Morris also retained a polling firm to test the popularity of specific arguments against ingredient disclosure and developed a strategic plan for opposing similar regulations in Vermont.

Conclusion

Tobacco companies have historically used third parties to form coalitions to oppose ingredient disclosure regulations. These coalitions have had success preventing regulations from being implemented after they are initially proposed by creating the appearance of local opposition. With countries around the world currently implementing ingredient disclosure regulations in the WHO Framework Convention on Tobacco, governments and regulatory agencies should be aware of the political strategies that the tobacco companies have used to create the impression of popular opposition to these measures.     

Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites'' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.     

Evolution and biogeography of the endemic Roucela complex (Campanulaceae: Campanula) in the Eastern Mediterranean

At the intersection of geological activity, climatic fluctuations, and human pressure, the Mediterranean Basin – a hotspot of biodiversity – provides an ideal setting for studying endemism, evolution, and biogeography. Here, we focus on the Roucela complex (Campanula subgenus Roucela), a group of 13 bellflower species found primarily in the eastern Mediterranean Basin. Plastid and low‐copy nuclear markers were employed to reconstruct evolutionary relationships and estimate divergence times within the Roucela complex using both concatenation and species tree analyses. Niche modeling, ancestral range estimation, and diversification analyses were conducted to provide further insights into patterns of endemism and diversification through time. Diversification of the Roucela clade appears to have been primarily the result of vicariance driven by the breakup of an ancient landmass. We found geologic events such as the formation of the mid‐Aegean trench and the Messinian Salinity Crisis to be historically important in the evolutionary history of this group. Contrary to numerous past studies, the onset of the Mediterranean climate has not promoted diversification in the Roucela complex and, in fact, may be negatively affecting these species. This study highlights the diversity and complexity of historical processes driving plant evolution in the Mediterranean Basin.