Miniprimer PCR, a New Lens for Viewing the Microbial World

Molecular methods based on the 16S rRNA gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. Here we show that a new PCR method using an engineered polymerase and 10-nucleotide “miniprimers” expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase. After testing the method in silico to identify divergent ribosomal genes in previously cloned environmental sequences, we applied the method to soil and microbial mat samples, which revealed novel 16S rRNA gene sequences that would not have been detected with standard primers. Deeply divergent sequences were discovered with high frequency and included representatives that define two new division-level taxa, designated CR1 and CR2, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.     

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): Effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T₁₄ and T₂₈, while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T₂₈ only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

Hunting and Plant Community Dynamics in Tropical Forests: A Synthesis and Future Directions

This synthesis builds on the preceding articles of this Special Section and has three goals. We first review the nascent literature that addresses indirect effects of hunting for tropical forest plant communities. Next, we highlight the potential indirect effects of hunting for other groups of organisms. Our final goal is to consider what could be done to ameliorate the demographic threats to harvest-sensitive game species caused by unsustainable hunting. Three conclusions are possible at this time concerning the impact of hunting for tropical forest plant communities: (1) Hunting tends to reduce seed movement for animal-dispersed species with very large diaspores; (2) Hunting reduces seed predation by granivorous vertebrates for species with large seeds; and (3) Hunting alters the species composition of the seedling and sapling layers. The cascading effects of hunting are already known to affect bruchid beetles and dung beetles and are likely to affect other, nongame taxa. To ameliorate these problems, several lines of research should be further explored to facilitate the development of game management plans including: (1) alternative use of sources of animal protein; (2) income supplementation for local people from sources other than wild meat; (3) outreach and extension activities for communities; (4) recognition and facilitation of the shifting of attitudes towards hunting; (5) implementation of community-based wildlife management programs in regulated-use areas such as extractive reserves; and (6) landscape-scale conservation planning that maximizes the source-sink dynamics of harvested and unharvested game populations and enforces game regulations in strictly protected areas.     

Limits to elevational distributions in two species of emberizine finches: disentangling the role of interspecific competition, autoecology, and geographic variation in the environment

When species' elevational ranges are wider where putative competitors are absent, researchers have concluded that interspecific competition influences elevational distributions. This overlooks the distinction between factors that limit distributions directly and factors that only influence organisms indirectly through covarying regulators or resources. Because elevation affects organisms indirectly, testing whether competition influences elevational ranges relies on the heretofore untested assumption that the relationship between elevation and factors influencing organisms directly is similar across geography. Focusing on Buarremon brush-finches (Aves: Emberizidae), a group in which distributions represent one of the best examples of the potential role of competition limiting elevational ranges, we show that when distributions are compared along axes of climatic variation, some patterns of elevational range variation do appear to be consistent with predictions of the hypothesis that release from competition underlies expanded elevational ranges in allopatry. However, other patterns of expanded elevational ranges in the absence of putative competitors are better explained by hypothesis related to species' autoecology and geographic variation in the environment. This latter finding cautions against using elevation uncritically as a dimension of ecological niches, and suggests that classical examples of interspecific competition may need re-evaluation.     

The sterol transporting heterodimer ABCG5/ABCG8 requires bile salts to mediate cholesterol efflux

The ATP binding cassette transporters ABCG5 and ABCG8 are indispensable for hepatobiliary cholesterol transport. In this study, we investigated the specificity of the heterodimer for cholesterol acceptors. Dog gallbladder epithelial cells were mono- or double-transfected with lentiviral mouse Abcg5 and Abcg8 vectors. Double-transfected cells showed increased efflux to different bile salt (BS) species, while mono-transfected cells did not show enhanced efflux. The efflux was initiated at micellar concentrations and addition of phosphatidylcholine increased efflux. Cholesterol secretion was highly BS dependent, whereas other cholesterol acceptors such as ApoAI, HDL or methyl-beta-cyclodextrin did not elicit Abcg5/g8 dependent cholesterol secretion.     

A decreased mitochondrial DNA content is related to insulin resistance in adolescents

The aim of this study was to investigate whether mitochondrial DNA (mtDNA) content is associated with insulin resistance (IR) in a sample of adolescents with features of metabolic syndrome. We further studied the link between polymorphisms in three genes involved in mitochondrial biogenesis and the presence of deleted mtDNA and mtDNA content. Data and blood samples were collected from 175 adolescents out of a cross-sectional, population-based study of 934 high school students. On the basis of the median value of homeostasis model assessment of IR (HOMA-IR) of the whole sample (2.2), the population was divided into two groups: noninsulin resistance (NIR) and IR. mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using a real-time quantitative PCR method. Genotyping for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) (pro12Ala), PPAR- gamma coactivator-1alpha (PGC-1alpha) (Gly482Ser), and Tfam (rs1937 and rs12247015) polymorphisms was performed by PCR-based restriction fragment length polymorphism. Long-extension PCR was performed to amplify the whole mitochondrial genome. The mtDNA/nDNA ratio was significantly lower in the IR group (median: 9.08, range: 68.94) in comparison with the NIR group (12.24, 71.92) (P<0.03). Besides, the mtDNA/nDNA ratio was inversely correlated with HOMA (R: -0.18, P<0.02), glucose (R: -0.21, P<0.008), and uric acid (R: -0.18, P<0.03). Genotypes for the PPAR- gamma, PGC-1alpha, and Tfam variants were not associated with the mtDNA/nDNA ratio. Long-extension PCR did not show significant levels of mtDNA deletions. In conclusion, our findings indicate that reduced mtDNA content in peripheral leukocytes is associated with IR. This result seems not to be related with the previously mentioned variants in genes involved in the regulation of mitochondrial biogenesis.     

Development of ChillPeach genomic tools and identification of cold-responsive genes in peach fruit

The ChillPeach database was developed to facilitate identification of genes controlling chilling injury (CI), a global-scale post-harvest physiological disorder in peach. It contained 7,862 high-quality ESTs (comprising 4,468 unigenes) obtained from mesocarp tissues of two full-sib progeny contrasting for CI, about 48 and 13% of which are unique to Prunus and Arabidopsis, respectively. All ESTs are in the Gateway vector to facilitate functional assessment of the genes. The data set contained several putative SNPs and 184 unigenes with high quality SSRs, of which 42% were novel to Prunus. Microarray slides containing 4,261 ChillPeach unigenes were printed and used in a pilot experiment to identify differentially expressed genes in cold-treated compared to control mesocarp tissues, and in vegetative compared to mesocarp tissues. Quantitative RT-PCR (qRT-PCR) confirmed microarray results for all 13 genes tested. The microarray and qRT-PCR analyses indicated that ChillPeach is rich in putative fruit-specific and novel cold-induced genes. A website ( http://bioinfo.ibmcp.upv.es/genomics/ChillPeachDB ) was created holding detailed information on the ChillPeach database.     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.