Molecular comparisons of the Culex pipiens (L.) complex esterase gene amplicons

The amplification of carboxylesterase genes is a mechanism of organophosphate resistance in Culex mosquitoes. Amplified carboxylesterase genes from an insecticide resistant Culex pipiens strain collected in Cyprus were analysed and compared to other Culex amplified carboxylesterase alleles. A 12 kb section of genomic DNA containing two gene loci coding for carboxylesterase alleles A5 and B5 was cloned and sequenced. A comparison between this amplicon and one from a strain with co-amplified carboxylesterase alleles A2 and B2 revealed a number of differences. The intergenic spacer was 3.7 kb in length in the A5-B5 amplicon (2.7 kb in A2-B2) and contained putative Juan and transposable elements upstream of B5. A fragment of a gene with high homology to aldehyde oxidase was also present immediately downstream of A5. The comparison revealed no differences that would explain the successful spread of the A2-B2 amplicon worldwide whilst the A5-B5 amplicon is restricted to the Mediterranean. (C) 2004 Elsevier Ltd. All rights reserved.

Longitudinal selenium status in healthy British adults: assessment using biochemical and molecular biomarkers

Human selenium (Se) requirements are currently based on biochemical markers of Se status. In rats, tissue glutathione peroxidase-1 (Gpx1) mRNA levels can be used effectively to determine Se requirements; blood Gpx1 mRNA levels decrease in Se-deficient rats, so molecular biology-based markers have potential for human nutrition assessment. To study the efficacy of molecular biology markers for assessing Se status in humans, we conducted a longitudinal study on 39 subjects (age 45 +/- 11) in Reading, UK. Diet diaries (5 day) and blood were obtained from each subject at 2, 8, 17 and 23 weeks, and plasma Se, glutathione peroxidase (Gpx3) enzyme activity, and selenoprotein mRNA levels were determined. There were no significant longitudinal effects on Se biomarkers. Se intake averaged 48 +/- 14 mu g/d. Plasma Se concentrations averaged 1.13 +/- 0.16 mu mol/l. Plasma Se v. energy-corrected Se intake (ng Se/kJ/d) was significantly correlated, but neither Gpx3 activity v. Se intake (ng Se/kJ/d) nor Gpx3 activity v. plasma Se was significantly correlated. Collectively, this indicates that subjects were on the plateaus of the response curves. Selenoprotein mRNAs were quantitated in total RNA isolated from whole blood, but mRNA levels for Gpx1, selenoprotein H, and selenoprotein W (all highly regulated by Se in rodents), as well selenoprotein P, Gpx3, and phospholipid hydroperoxide glutathione peroxidase were also not significantly correlated with plasma Se. Thus selenoprotein molecular biomarkers, as well as traditional biochemical markers, are unable to further distinguish differences in Se status in these Se replete subjects. The efficacy of molecular biomarkers to detect Se deficiency needs to be tested in Se-deficient populations.

SARS coronavirus unique domain: three-domain molecular architecture in solution and RNA binding

Nonstructural protein 3 of the severe acute respiratory syndrome (SARS) coronavirus includes a "SARS-unique domain" (SUD) consisting of three globular domains separated by short linker peptide segments. This work reports NMR structure determinations of the C-terminal domain (SUD-C) and a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution; in SUD-NM, there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel-shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observations of (15)N-labeled proteins further resulted in delineation of RNA binding sites (i.e., in SUD-M, a positively charged surface area with a pronounced cavity, and in SUD-C, several residues of an anti-parallel beta-sheet). Overall, the present data provide evidence for molecular mechanisms involving the concerted actions of SUD-M and SUD-C, which result in specific RNA binding that might be unique to the SUD and, thus, to the SARS coronavirus.

Application of molecular biological methods for studying probiotics and the gut flora

Increasingly, the microbiological scientific community is relying on molecular biology to define the complexity of the gut flora and to distinguish one organism from the next. This is particularly pertinent in the field of probiotics, and probiotic therapy, where identifying probiotics from the commensal flora is often warranted. Current techniques, including genetic fingerprinting, gene sequencing, oligonucleotide probes and specific primer selection, discriminate closely related bacteria with varying degrees of success. Additional molecular methods being employed to determine the constituents of complex microbiota in this area of research are community analysis, denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE), fluorescent in situ hybridisation (FISH) and probe grids. Certain approaches enable specific aetiological agents to be monitored, whereas others allow the effects of dietary intervention on bacterial populations to be studied. Other approaches demonstrate diversity, but may not always enable quantification of the population. At the heart of current molecular methods is sequence information gathered from culturable organisms. However, the diversity and novelty identified when applying these methods to the gut microflora demonstrates how little is known about this ecosystem. Of greater concern is the inherent bias associated with some molecular methods. As we understand more of the complexity and dynamics of this diverse microbiota we will be in a position to develop more robust molecular-based technologies to examine it. In addition to identification of the microbiota and discrimination of probiotic strains from commensal organisms, the future of molecular biology in the field of probiotics and the gut flora will, no doubt, stretch to investigations of functionality and activity of the microflora, and/or specific fractions. The quest will be to demonstrate the roles of probiotic strains in vivo and not simply their presence or absence.

Molecular mechanisms of oestrogen action on growth of human breast cancer cells in culture

Growth responses to oestrogen can be reproducibly obtained using a selection of oestrogen-receptor-containing human breast cancer cell lines, and molecular mechanisms have been shown to include modulation to growth factor/receptor/signalling pathways, cell-cycle proteins, apoptosis, differentiation, adhesion, motility and migration. Considerable progress has been made in understanding the molecular basis of oestrogen action on gene expression through the ligand-activated transcription factors human oestrogen receptor α (ERα) and ERβ and the resulting effects on global gene expression patterns, but the full profile of coordination of the alterations, which brings about changes in cell growth through genomic and non-genomic mechanisms remain to be fully elucidated. Oestrogen regulation of cell growth involves a complex cross-talk between oestrogen receptor and growth factor signalling pathways such that inhibition of one pathway may lead to stimulation of another, which may explain the remarkable ability of human breast cancer cells to escape from any mode of imposed growth inhibition be it oestrogen deprivation or administration of antioestrogen. Although studies on cell growth have focused to date on the effects of physiological oestrogens, many hundreds of environmental chemicals with oestrogenic properties have now been measured in the human breast. Whether or not the weight of evidence eventually establishes any causal link of complex mixtures of environmental oestrogenic chemicals with breast cancer, the presence of so many oestrogenic chemicals in the breast must influence resulting oestrogenic responses, and the impact of this additional oestrogenic burden needs to be taken into account in future studies on growth regulation of human breast cancer cells.

Molecular insights of p47phox phosphorylation dynamics in the regulation of NADPH oxidase activation and superoxide production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47phox_/_ coronary microvascular cells. Compared with wild-type p47phoxcDNAtransfected cells, the single mutation of S379A completely blocked p47phox membrane translocation, binding to p22phox and endothelial O2 . production in response to acute stimulation of PKC. p47phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47phox conformational changes and NADPH oxidase-dependent superoxide production by cells.

Forest-obligate Sabethes mosquitoes suggest palaeoecological perturbations

The origin of tropical forest diversity has been hotly debated for decades. Although specific mechanisms vary, many such explanations propose some vicariance in the distribution of species during glacial cycles and several have been supported by genetic evidence in Neotropical taxa. However, no consensus exists with regard to the extent or time frame of the vicariance events. Here, we analyse the cytochrome oxidase II mitochondrial gene of 250 Sabethes albiprivus B mosquitoes sampled from western Sao Paulo in Brazil. There was very low population structuring among collection sites (Phi(ST) = 0.03, P = 0.04). Historic demographic analyses and the contemporary geographic distribution of genetic diversity suggest that the populations sampled are not at demographic equilibrium. Three distinct mitochondrial clades were observed in the samples, one of which differed significantly in its geographic distribution relative to the other two within a small sampling area (similar to 70 x 35 km). This fact, supported by the inability of maximum likelihood analyses to achieve adequate fits to simple models for the population demography of the species, suggests a more complex history, possibly involving disjunct forest refugia. This hypothesis is supported by a genetic signal of recent population growth, which is expected if population sizes of this forest-obligate insect increased during the forest expansions that followed glacial periods. Although a time frame cannot be reliably inferred for the vicariance event leading to the three genetic clades, molecular clock estimates place this at similar to 1 Myr before present.

Copia Retrotransposon in the Zaprionus Genus: Another Case of Transposable Element Sharing with the Drosophila melanogaster Subgroup

Copia is a retrotransposon that appears to be distributed widely among the Drosophilidae subfamily. Evolutionary analyses of regulatory regions have indicated that the Copia retrotransposon evolved through both positive and purifying selection, and that horizontal transfer (HT) could also explain its patchy distribution of the among the subfamilies of the melanogaster subgroup. Additionally, Copia elements could also have transferred between melanogaster subgroup and other species of Drosophilidae-D. willistoni and Z. tuberculatus. In this study, we surveyed seven species of the Zaprionus genus by sequencing the LTR-ULR and reverse transcriptase regions, and by using RT-PCR in order to understand the distribution and evolutionary history of Copia in the Zaprionus genus. The Copia element was detected, and was transcriptionally active, in all species investigated. Structural and selection analysis revealed Zaprionus elements to be closely related to the most ancient subfamily of the melanogaster subgroup, and they seem to be evolving mainly under relaxed purifying selection. Taken together, these results allowed us to classify the Zaprionus sequences as a new subfamily-ZapCopia, a member of the Copia retrotransposon family of the melanogaster subgroup. These findings indicate that the Copia retrotransposon is an ancient component of the genomes of the Zaprionus species and broaden our understanding of the diversity of retrotransposons in the Zaprionus genus.

EVOLUTIONARY BIOLOGY IN BIODIVERSITY SCIENCE, CONSERVATION, AND POLICY: A CALL TO ACTION

Evolutionary biologists have long endeavored to document how many species exist on Earth, to understand the processes by which biodiversity waxes and wanes, to document and interpret spatial patterns of biodiversity, and to infer evolutionary relationships. Despite the great potential of this knowledge to improve biodiversity science, conservation, and policy, evolutionary biologists have generally devoted limited attention to these broader implications. Likewise, many workers in biodiversity science have underappreciated the fundamental relevance of evolutionary biology. The aim of this article is to summarize and illustrate some ways in which evolutionary biology is directly relevant We do so in the context of four broad areas: (1) discovering and documenting biodiversity, (2) understanding the causes of diversification, (3) evaluating evolutionary responses to human disturbances, and (4) implications for ecological communities, ecosystems, and humans We also introduce bioGENESIS, a new project within DIVERSITAS launched to explore the potential practical contributions of evolutionary biology In addition to fostering the integration of evolutionary thinking into biodiversity science, bioGENESIS provides practical recommendations to policy makers for incorporating evolutionary perspectives into biodiversity agendas and conservation. We solicit your involvement in developing innovative ways of using evolutionary biology to better comprehend and stem the loss of biodiversity.

Generic relationships and dating of lineages in Winteraceae based on nuclear (ITS) and plastid (rpS16 and psbA-trnH) sequence data

Phylogenetic analyses of representative species from the five genera of Winteraceae (Drimys, Pseudowintera, Takhtajania, Tasmannia, and Zygogynum s.l.) were performed using ITS nuclear sequences and a combined data-set of ITS + psbA-trnH + rpS16 sequences (sampling of 30 and 15 species, respectively). Indel informativity using simple gap coding or gaps as a fifth character was examined in both data-sets. Parsimony and Bayesian analyses support the monophyly of Drimys, Tasmannia, and Zygogynum s.l., but do not support the monophyly of Belliolum, Zygogynum s.s., and Bubbia. Within Drimys, the combined data-set recovers two subclades. Divergence time estimates suggest that the splitting between Drimys and its sister clade (Pseudowintera + Zygogynum s.l.) occurred around the end of the Cretaceous; in contrast, the divergence between the two subclades within Drimys is more recent (15.5-18.5 MY) and coincides in time with the Andean uplift. Estimates suggest that the earliest divergences within Winteraceae could have predated the first events of Gondwana fragmentation. (C) 2009 Elsevier Inc. All rights reserved.

Phylogeny, species limits, and biogeography of the Brazilian lizards of the genus Eurolophosaurus (Squamata : Tropiduridae) as inferred from mitochondrial DNA sequences

Phylogenetic relationships and divergence times for 10 populations of the three recognized ""species"" of Brazilian lizards of genus Eurolophosaurus were estimated from 1229 bp of cyt b, COI, 12S, and 16S rRNA mitochondrial gene segments. Eurolophosaurus is monophyletic and the basal split within the genus separates E divaricatus from a clade comprising E amathites and E nanuzae. Three populations of E divaricatus, which occurs along the western bank of Rio S (a) over tildeo Francisco, were consistently grouped together. Oil the east bank of the river, E amathites and E nanuzae from state of Bahia were recovered as the sister group of E nanuzae populations from state of Minas Gerais. The paraphyly of E nanuzae and the high divergence levels among populations of E divaricatus strongly suggest that species limits in Eurolophosaurus should be revised. Even considering an extreme evolutionary rate of 2.8% sequence divergence per million years for the four gene segments analyzed together, E. divaricatus would have separated from the two other species by at least 5.5 my ago, and E. amathites from E nanuzae populations from Bahia and Minas Gerais, respectively, by 1.5 and 3.5 my. The paleolacustrine hypothesis and changes in the course of the river potentially explain faunal divergence in the area, but divergences are much older than previously admitted. (C) 2007 Elsevier Inc. All rights reserved.

Independent fusions and recent origins of sex chromosomes in the evolution and diversification of glass knife fishes (Eigenmannia)

The genus Eigenmannia comprises several species groups that display a surprising variety of diploid chromosome numbers and sex-determining systems. In this study, hypotheses regarding phylogenetic relationships and karyotype evolution were investigated using a combination of molecular and cytogenetic methods. Phylogenetic relationships were analyzed for 11 cytotypes based on sequences from five mitochondrial DNA regions. Parsimony-based character mapping of sex chromosomes confirms previous suggestions of multiple origins of sex chromosomes. Molecular cytogenetic analyses involved chromosome painting using probes derived from whole sex chromosomes from two taxa that were hybridized to metaphases of their respective sister cytotypes. These analyses showed that a multiple XY system evolved recently (<7 mya) by fusion. Furthermore, one of the chromosomes that fused to form the neo-Y chromosome is fused independently to another chromosome in the sister cytotype. This may constitute an efficient post-mating barrier and might imply a direct function of sex chromosomes in the speciation processes in Eigenmannia. The other chromosomal sex-determination system investigated is shown to have differentiated by an accumulation of heterochromatin on the X chromosome. This has occurred in the past 0.6 my, and is the most recent chromosomal sex-determining system described to date. These results show that the evolution of sex-determining systems can proceed very rapidly. Heredity (2011) 106, 391-400; doi:10.1038/hdy.2010.82; published online 23 June 2010

Genetic structure, phylogeny, and biogeography of Brazilian eyelid-less lizards of genera Calyptommatus and Nothobachia (Squamata, Gymnophthalmidae) as inferred from mitochondrial DNA sequences

Calyptommatus and Nothobachia genera of gymnophthalmid lizards are restricted to sandy open habitats on Sao Francisco River margins, northeastern Brazil. Phylogenetic relationships and geographic distribution of the four recognized species of Calyptommatus were analyzed from partial mitochondrial cyt b, 12S, and 16S rRNA genes sequencing, taking allopatric populations of the monotypic Nothobachia ablephara as the outgroup. In Calyptommatus a basal split separated C. sinebrachiatus, a species restricted to the eastern bank of the river, from the three other species. In this clade, C. confusionibus, found on western margin, was recovered as the sister group of the two other species, C. leiolepis and C. nicterus, from opposite margins. According to approximate date estimations, C. sinebrachiatus would have separated from the other congeneric species by 4.4-6.5 my, and C. nicterus, also from eastern bank, would be diverging by 1.8-2.6 my from C. leiolepis, the sister species on the opposite margin. C. confusionibus and C. leiolepis, both from western sandy areas, would be differentiating by 2.8-5.0 my. Divergence times of about 3.0-4.0 my were estimated for allopatric populations of Nothobachia restricted to western margin. Significant differences in 16S rRNA secondary structure relatively to other vertebrates are reported. Distinct evolutionary patterns are proposed for different taxa in those sandy areas, probably related to historical changes in the course of Sao Francisco River. (C) 2010 Elsevier Inc. All rights reserved.

Towards an assessment of character interdependence in avian RNA phylogenetics: A general secondary structure model for the avian mitochondrial 16S rRNA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The timing of Neotropical speciation dynamics: A reconstruction of Myiopagis flycatcher diversification using phylogenetic and paleogeographic data

Neotropical forests have brought forth a large proportion of the world`s terrestrial biodiversity, but the underlying evolutionary mechanisms and their timing require further elucidation. Despite insights gained from phylogenetic studies, uncertainties about molecular clock rates have hindered efforts to determine the timing of diversification processes. Moreover, most molecular research has been detached from the extensive body of data on Neotropical geology and paleogeography. We here examine phylogenetic relationships and the timing of speciation events in a Neotropical flycatcher genus (Myiopagis) by using calibrations from modern geologic data in conjunction with a number of recently developed DNA sequence dating algorithms and by comparing these estimates with those based on a range of previously proposed molecular clock rates. We present a well-supported hypothesis of systematic relationships within the genus. Our age estimates of Myiopagis speciation events based on paleogeographic data are in close agreement with nodal ages derived from a ""traditional"" avian mitochondrial 2%/My clock, while contradicting other clock rates. Our comparative approach corroborates the consistency of the traditional avian mitochondrial clock rate of 2%/My for tyrant-flycatchers. Nevertheless, our results argue against the indiscriminate use of molecular clock rates in evolutionary research and advocate the verification of the appropriateness of the traditional clock rate by means of independent calibrations in individual studies. (C) 2009 Elsevier Inc. All rights reserved.