Widespread convergence in toxin resistance by predictable molecular evolution

The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na(+)/K(+)-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na(+)/K(+)-ATPase H1-H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na(+)/K(+)-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.

Functional analysis of yeast snoRNA and snRNA 3' end formation mediated by uncoupling of cleavage and polyadenylation

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3′-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3′ portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Dimerization of retroviral RNA genomes : an inseparable pair

Many viruses carry more than one segment of nucleic acid into the virion particle, but retroviruses are the only known group of viruses that contain two identical (or nearly identical) copies of the RNA genome within the virion. These RNA genomes are non-covalently joined together through a process known as genomic RNA dimerization. Uniquely, the RNA dimerization of the retroviral genome is of crucial importance for efficient retroviral replication. In this article, our current understanding of the relationship between retroviral genome conformation, dimerization and replication is reviewed.

Mitochondrial DNA analysis of field populations of Helicoverpa armigera (Lepidoptera : Noctuidae) and of its relationship to H. zea

Background
Helicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea.

Results
We obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017 – 0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago.

Conclusion
Our mitochondrial DNA sequence data supports the single species status of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either.

Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

Swine influenza virus PA and neuraminidase gene reassortment into human H1N1iInfluenza Virus is associated with an altered pathogenic phenotype linked to increased MIP-2 expression

Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. IMPORTANCE: Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Lipids in Amyloid-β processing, aggregation, and toxicity

Aggregation of amyloid-beta (Aβ) peptide is the major event underlying neuronal damage in Alzheimer's disease (AD). Specific lipids and their homeostasis play important roles in this and other neurodegenerative disorders. The complex interplay between the lipids and the generation, clearance or deposition of Aβ has been intensively investigated and is reviewed in this chapter. Membrane lipids can have an important influence on the biogenesis of Aβ from its precursor protein. In particular, increased cholesterol in the plasma membrane augments Aβ generation and shows a strong positive correlation with AD progression. Furthermore, apolipoprotein E, which transports cholesterol in the cerebrospinal fluid and is known to interact with Aβ or compete with it for the lipoprotein receptor binding, significantly influences Aβ clearance in an isoform-specific manner and is the major genetic risk factor for AD. Aβ is an amphiphilic peptide that interacts with various lipids, proteins and their assemblies, which can lead to variation in Aβ aggregation in vitro and in vivo. Upon interaction with the lipid raft components, such as cholesterol, gangliosides and phospholipids, Aβ can aggregate on the cell membrane and thereby disrupt it, perhaps by forming channel-like pores. This leads to perturbed cellular calcium homeostasis, suggesting that Aβ-lipid interactions at the cell membrane probably trigger the neurotoxic cascade in AD. Here, we overview the roles of specific lipids, lipid assemblies and apolipoprotein E in Aβ processing, clearance and aggregation, and discuss the contribution of these factors to the neurotoxicity in AD.

The use of molecular phylogenetic and morphological tools to identify cryptic and paraphyletic species : examples from the diminutive long-fingered bats (Chiroptera : Miniopteridae : Miniopterus) on Madagascar

Based on nearly complete (1125 bp) cytochrome-b sequence data and morphological characters, two new endemic species of Miniopterus are described from Madagascar that were previously identified as M. manavi. Using phylogenetic analysis, the basal nodes of major lineages in the Malagasy members of this genus are weakly supported, while, in most cases, the branches leading to each of the clades are well resolved. Miniopterus mahafaliensis, new species, occurs in the southwestern semidesert areas and M. brachytragos, new species, has a broad distribution across the northern half of the island, ranging across several different biomes. Phylogenetic inference indicates that these two new taxa are not closely related to M. manavi sensu stricto, with average genetic distances of 9.2% and 5.7% from this taxon, respectively. On the basis of this and previous revisions, the former M. manavi complex is now recognized to represent at least five taxa, which do not form a monophyletic group with respect to one another, and represent extraordinary examples of convergent evolution. Miniopterus brachytragos is closely related to the recently named M. aelleni, while M. mahafaliensis is not closely associated with any of these species. Molecular phylogenetic analysis was imperative to resolve the species limits of these taxa and morphology then provided the means to corroborate the recovered clades. There are localities on the island, specifically limestone karstic zones, where four species of the former M. manavi sensu lato complex occur in strict sympatry. These species often use the same day-roost caves and have similar external and craniodental measurements. This raises intriguing questions as to how these animals divide their worlds with regard to dietary regimes and foraging strategies, as well as their speciation history.

The use of molecular and morphological characters to resolve the taxonomic identity of cryptic species : the case of Miniopterus manavi (Chiroptera, Miniopteridae)

Based on recent molecular phylogenetic studies, the Old World bat family Miniopteridae, composed of species in the genus Miniopterus, has been shown to contain complex paraphyletic species, many of which are cryptic based on convergent morphological characters. Herein we resolve the phylogenetic relationships and taxonomy of the species complex M. manavi on Madagascar and in the Comoro Archipelago, where these animals occur in different bioclimatic zones. First using mitochondrial cytochrome-b sequence data to define clades and then morphology to corroborate the molecular data, including comparisons to type specimens, we demonstrate that animals identified as this taxon are a minimum of three species: M. manavi sensu stricto occurs in at least the central portion of the Central Highlands; M. griveaudi has a broad distribution in lowland northern and central western Madagascar and the Comoros (Anjouan and Grande Comore), and M. aelleni sp. n. has been found in northern and western Madagascar and the Comoros (Anjouan). In each case, these three clades were genetically divergent and monophyletic and the taxa are diagnosable based on different external and craniodental characters. One aspect that helped to define the systematics of this group was isolation of DNA from one of the paratypes of M. manavi collected in 1896 and new topotypic material. Miniopterus manavi is most closely allied to a recently described species, M. petersoni. At several localities, M. griveaudi and M. aelleni have been found in strict sympatry, and together with M. manavi sensu stricto show considerable convergence in morphological characters, but are not immediate sister taxa. In defining and resolving the systematics of cryptic species, such as miniopterid bats, the process of defining clades with molecular tools, segregating the specimens accordingly, and identifying corroborative morphological characters has been notably efficient.

Non-referenced genome assembly from epigenomic short-read data

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.

Molecular genetic variation in Galaxias maculatus (Osteichthys: Galaxiidae)

The "common jollytail", Galaxias maculatus, is a freshwater minnow-like fish that is distributed across the Southern Hemisphere and occurs abundantly in inland waterways of south-eastern Australia. Molecular genetic data were collected from samples of this species throughout western Victoria to evaluate hypotheses concerning the taxonomy and evolution of this ecologically important fish.

Obtaining accurate frequencies of sequential patterns over a single sequence

In the mining and analysis of a single long sequence, one fundamental and important problem is obtaining accurate frequencies of sequential patterns over the sequence. However, we identify that five previous frequency measures suffer from inherent inaccuracies. To obtain more accurate frequencies, we introduce two basic principles called strict anti-monotonicity and maximum-count for frequency measures. Under the two principles, a new frequency measure is presented. An algorithm is also devised to compute it. Both theoretical analysis and empirical evaluation show that more accurate frequencies can be obtained under the new measure

The role of the ord arid intrusion in the historical and contemporary genetic division of long-tailed finch subspecies in northern Australia

The effect of separation by biogeographic features followed by secondary contact can blur taxonomic boundaries and produce complex genetic signatures. We analyzed population structure and gene flow across the range of the long-tailed finch (Poephila acuticauda) in northern Australia (1) to test the hypothesis that Ord Arid Intrusion acted as the causative barrier that led to divergence of P. acuticauda subspecies, (2) to determine whether genetic data support the presence of a gradual cline across the range or a sudden shift, both of which have been suggested based on morphological data, and (3) to estimate levels of contemporary gene flow within this species complex. We collected samples from 302 individuals from 10 localities. Analyses of 12 microsatellite loci and sequence data from 333 base pairs of the mitochondrial control region were used to estimate population structure and gene flow, using analysis of molecular variance (AMOVA), haplotype network analysis, frequency statistics, and clustering methods. Mitochondrial sequence data indicated the presence of three genetic groups (regions) across the range of P. acuticauda. Genetic diversity was highest in the east and lowest in the west. The Ord Arid Intrusion appears to have functioned as a biogeographic barrier in the past, according to mtDNA evidence presented here and evidence from previous studies. The absence of isolation by distance between adjacent regions and the lack of population genetic structure of mtDNA within regions indicates that genetic changes across the range of P. acuticauda subspecies are characterized by discrete breaks between regions. While microsatellite data indicate a complete absence of genetic structure across this species’ range, it appears unlikely that this results from high levels of gene flow. Mitochondrial data do not support the presence of contemporary gene flow across the range of this species.

Effects of Mitochondrial DNA Rate Variation on Reconstruction of Pleistocene Demographic History in a Social Avian Species, Pomatostomus superciliosus

Mitochondrial sequence data is often used to reconstruct the demographic history of Pleistocene populations in an effort to understand how species have responded to past climate change events. However, departures from neutral equilibrium conditions can confound evolutionary inference in species with structured populations or those that have experienced periods of population expansion or decline. Selection can affect patterns of mitochondrial DNA variation and variable mutation rates among mitochondrial genes can compromise inferences drawn from single markers. We investigated the contribution of these factors to patterns of mitochondrial variation and estimates of time to most recent common ancestor (TMRCA) for two clades in a co-operatively breeding avian species, the white-browed babbler Pomatostomus superciliosus. Both the protein-coding ND3 gene and hypervariable domain I control region sequences showed departures from neutral expectations within the superciliosus clade, and a two-fold difference in TMRCA estimates. Bayesian phylogenetic analysis provided evidence of departure from a strict clock model of molecular evolution in domain I, leading to an over-estimation of TMRCA for the superciliosus clade at this marker. Our results suggest mitochondrial studies that attempt to reconstruct Pleistocene demographic histories should rigorously evaluate data for departures from neutral equilibrium expectations, including variation in evolutionary rates across multiple markers. Failure to do so can lead to serious errors in the estimation of evolutionary parameters and subsequent demographic inferences concerning the role of climate as a driver of evolutionary change. These effects may be especially pronounced in species with complex social structures occupying heterogeneous environments. We propose that environmentally driven differences in social structure may explain observed differences in evolutionary rate of domain I sequences, resulting from longer than expected retention times for matriarchal lineages in the superciliosus clade.

Strong genetic structure corresponds to small-scale geographic breaks in the Australian alpine grasshopper Kosciuscola tristis

BACKGROUND: Mountain landscapes are topographically complex, creating discontinuous 'islands' of alpine and sub-alpine habitat with a dynamic history. Changing climatic conditions drive their expansion and contraction, leaving signatures on the genetic structure of their flora and fauna. Australia's high country covers a small, highly fragmented area. Although the area is thought to have experienced periods of relative continuity during Pleistocene glacial periods, small-scale studies suggest deep lineage divergence across low-elevation gaps. Using both DNA sequence data and microsatellite markers, we tested the hypothesis that genetic partitioning reflects observable geographic structuring across Australia's mainland high country, in the widespread alpine grasshopper Kosciuscola tristis (Sjösted). RESULTS: We found broadly congruent patterns of regional structure between the DNA sequence and microsatellite datasets, corresponding to strong divergence among isolated mountain regions. Small and isolated mountains in the south of the range were particularly distinct, with well-supported divergence corresponding to climate cycles during the late Pliocene and Pleistocene. We found mixed support, however, for divergence among other mountain regions. Interestingly, within areas of largely contiguous alpine and sub-alpine habitat around Mt Kosciuszko, microsatellite data suggested significant population structure, accompanied by a strong signature of isolation-by-distance. CONCLUSIONS: Consistent patterns of strong lineage divergence among different molecular datasets indicate genetic breaks between populations inhabiting geographically distinct mountain regions. Three primary phylogeographic groups were evident in the highly fragmented Victorian high country, while within-region structure detected with microsatellites may reflect more recent population isolation. Despite the small area of Australia's alpine and sub-alpine habitats, their low topographic relief and lack of extensive glaciation, divergence among populations was on the same scale as that detected in much more extensive Northern hemisphere mountain systems. The processes driving divergence in the Australian mountains might therefore differ from their Northern hemisphere counterparts.