Expression and Purification of Recombinant Hemoglobin in Escherichia coli

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis

Contrasting Genetic Structure in Two Co-Distributed Species of Old World Fruit Bat

The fulvous fruit bat (Rousettus leschenaulti) and the greater short-nosed fruit bat (Cynopterus sphinx) are two abundant and widely co-distributed Old World fruit bats in Southeast and East Asia. The former species forms large colonies in caves while the latter roots in small groups in trees. To test whether these differences in social organization and roosting ecology are associated with contrasting patterns of gene flow, we used mtDNA and nuclear loci to characterize population genetic subdivision and phylogeographic histories in both species sampled from China, Vietnam and India. Our analyses from R. leschenaulti using both types of marker revealed little evidence of genetic structure across the study region. On the other hand, C. sphinx showed significant genetic mtDNA differentiation between the samples from India compared with China and Vietnam, as well as greater structuring of microsatellite genotypes within China. Demographic analyses indicated signatures of past rapid population expansion in both taxa, with more recent demographic growth in C. sphinx. Therefore, the relative genetic homogeneity in R. leschenaulti is unlikely to reflect past events. Instead we suggest that the absence of substructure in R. leschenaulti is a consequence of higher levels of gene flow among colonies, and that greater vagility in this species is an adaptation associated with cave roosting

A Bayesian Outlier Criterion to Detect SNPs under Selection in Large Data Sets

Background: The recent advent of high-throughput SNP genotyping technologies has opened new avenues of research for population genetics. In particular, a growing interest in the identification of footprints of selection, based on genome scans for adaptive differentiation, has emerged.[br/] Methodology/Principal Findings: The purpose of this study is to develop an efficient model-based approach to perform Bayesian exploratory analyses for adaptive differentiation in very large SNP data sets. The basic idea is to start with a very simple model for neutral loci that is easy to implement under a Bayesian framework and to identify selected loci as outliers via Posterior Predictive P-values (PPP-values). Applications of this strategy are considered using two different statistical models. The first one was initially interpreted in the context of populations evolving respectively under pure genetic drift from a common ancestral population while the second one relies on populations under migration-drift equilibrium. Robustness and power of the two resulting Bayesian model-based approaches to detect SNP under selection are further evaluated through extensive simulations. An application to a cattle data set is also provided.[br/] Conclusions/Significance: The procedure described turns out to be much faster than former Bayesian approaches and also reasonably efficient especially to detect loci under positive selection

BAC-Based Sequencing of Behaviorally-Relevant Genes in the Prairie Vole

The prairie vole (Microtus ochrogaster) is an important model organism for the study of social behavior, yet our ability to correlate genes and behavior in this species has been limited due to a lack of genetic and genomic resources. Here we report the BAC-based targeted sequencing of behaviorally-relevant genes and flanking regions in the prairie vole. A total of 6.4 Mb of non-redundant or haplotype-specific sequence assemblies were generated that span the partial or complete sequence of 21 behaviorally-relevant genes as well as an additional 55 flanking genes. Estimates of nucleotide diversity from 13 loci based on alignments of 1.7 Mb of haplotype-specific assemblies revealed an average pair-wise heterozygosity (8.4×10−3). Comparative analyses of the prairie vole proteins encoded by the behaviorally-relevant genes identified >100 substitutions specific to the prairie vole lineage. Finally, our sequencing data indicate that a duplication of the prairie vole AVPR1A locus likely originated from a recent segmental duplication spanning a minimum of 105 kb. In summary, the results of our study provide the genomic resources necessary for the molecular and genetic characterization of a high-priority set of candidate genes for regulating social behavior in the prairie vole

Phylogeny of Echinoderm Hemoglobins

Recent genomic information has revealed that neuroglobin and cytoglobin are the two principal lineages of vertebrate hemoglobins, with the latter encompassing the familiar myoglobin and α-globin/β-globin tetramer hemoglobin, and several minor groups. In contrast, very little is known about hemoglobins in echinoderms, a phylum of exclusively marine organisms closely related to vertebrates, beyond the presence of coelomic hemoglobins in sea cucumbers and brittle stars. We identified about 50 hemoglobins in sea urchin, starfish and sea cucumber genomes and transcriptomes, and used Bayesian inference to carry out a molecular phylogenetic analysis of their relationship to vertebrate sequences, specifically, to assess the hypothesis that the neuroglobin and cytoglobin lineages are also present in echinoderms.The genome of the sea urchin Strongylocentrotus purpuratus encodes several hemoglobins, including a unique chimeric 14-domain globin, 2 androglobin isoforms and a unique single androglobin domain protein. Other strongylocentrotid genomes appear to have similar repertoires of globin genes. We carried out molecular phylogenetic analyses of 52 hemoglobins identified in sea urchin, brittle star and sea cucumber genomes and transcriptomes, using different multiple sequence alignment methods coupled with Bayesian and maximum likelihood approaches. The results demonstrate that there are two major globin lineages in echinoderms, which are related to the vertebrate neuroglobin and cytoglobin lineages. Furthermore, the brittle star and sea cucumber coelomic hemoglobins appear to have evolved independently from the cytoglobin lineage, similar to the evolution of erythroid oxygen binding globins in cyclostomes and vertebrates.The presence of echinoderm globins related to the vertebrate neuroglobin and cytoglobin lineages suggests that the split between neuroglobins and cytoglobins occurred in the deuterostome ancestor shared by echinoderms and vertebrates

Drought-Adaptation Potential in Fagus sylvatica: Linking Moisture Availability with Genetic Diversity and Dendrochronology

<div><h3>Background</h3><p>Microevolution is essential for species persistence especially under anticipated climate change scenarios. Species distribution projection models suggested that the dominant tree species of lowland forests in Switzerland, European beech (<em>Fagus sylvatica</em> L.), might disappear from most areas due to expected longer dry periods. However, if genotypes at the moisture boundary of the species climatic envelope are adapted to lower moisture availability, they can serve as seed source for the continuation of beech forests under changing climates.</p> <h3>Methodology/Principal Findings</h3><p>With an AFLP genome scan approach, we studied neutral and potentially adaptive genetic variation in <em>Fagus sylvatica</em> in three regions containing a dry and a mesic site each (<em>n</em>_ind. = 241, <em>n</em>_markers = 517). We linked this dataset with dendrochronological growth measures and local moisture availabilities based on precipitation and soil characteristics. Genetic diversity decreased slightly at dry sites. Overall genetic differentiation was low (<em>F</em>_st = 0.028) and Bayesian cluster analysis grouped all populations together suggesting high (historical) gene flow. The Bayesian outlier analyses indicated 13 markers with three markers differing between all dry and mesic sites and the others between the contrasting sites within individual regions. A total of 41 markers, including seven outlier loci, changed their frequency with local moisture availability. Tree height and median basal growth increments were reduced at dry sites, but marker presence/absence was not related to dendrochronological characteristics.</p> <h3>Conclusion and Their Significance</h3><p>The outlier alleles and the makers with changing frequencies in relation to moisture availability indicate microevolutionary processes occurring within short geographic distances. The general genetic similarity among sites suggests that ‘preadaptive’ genes can easily spread across the landscape. Yet, due to the long live span of trees, fostering saplings originating from dry sites and grown within mesic sites might increase resistance of beech forests during the anticipated longer dry periods.</p> </div

Production of ultrasonic vocalizations by Peromyscus mice in the wild

BACKGROUND: There has been considerable research on rodent ultrasound in the laboratory and these sounds have been well quantified and characterized. Despite the value of research on ultrasound produced by mice in the lab, it is unclear if, and when, these sounds are produced in the wild, and how they function in natural habitats. RESULTS: We have made the first recordings of ultrasonic vocalizations produced by two free-living species of mice in the genus Peromyscus (P. californicus and P. boylii) on long term study grids in California. Over 6 nights, we recorded 65 unique ultrasonic vocalization phrases from Peromyscus. The ultrasonic vocalizations we recorded represent 7 different motifs. Within each motif, there was considerable variation in the acoustic characteristics suggesting individual and contextual variation in the production of ultrasound by these species. CONCLUSION: The discovery of the production of ultrasonic vocalizations by Peromyscus in the wild highlights an underappreciated component in the behavior of these model organisms. The ability to examine the production of ultrasonic vocalizations in the wild offers excellent opportunities to test hypotheses regarding the function of ultrasound produced by rodents in a natural context

Study of the reaction e^{+}e^{-} -->J/psi\pi^{+}\pi^{-} via initial-state radiation at BaBar

We study the process $e^+e^-\to J/\psi\pi^{+}\pi^{-}$ with initial-state-radiation events produced at the PEP-II asymmetric-energy collider. The data were recorded with the BaBar detector at center-of-mass energies 10.58 and 10.54 GeV, and correspond to an integrated luminosity of 454 $\mathrm{fb^{-1}}$. We investigate the $J/\psi \pi^{+}\pi^{-}$ mass distribution in the region from 3.5 to 5.5 $\mathrm{GeV/c^{2}}$. Below 3.7 $\mathrm{GeV/c^{2}}$ the $\psi(2S)$ signal dominates, and above 4 $\mathrm{GeV/c^{2}}$ there is a significant peak due to the Y(4260). A fit to the data in the range 3.74 -- 5.50 $\mathrm{GeV/c^{2}}$ yields a mass value $4244 \pm 5$ (stat) $\pm 4$ (syst)$\mathrm{MeV/c^{2}}$ and a width value $114 ^{+16}_{-15}$ (stat)$\pm 7$(syst)$\mathrm{MeV}$ for this state. We do not confirm the report from the Belle collaboration of a broad structure at 4.01 $\mathrm{GeV/c^{2}}$. In addition, we investigate the $\pi^{+}\pi^{-}$ system which results from Y(4260) decay

Evolutionary factors affecting Lactate dehydrogenase A and B variation in the Daphnia pulex species complex

Background: Evidence for historical, demographic and selective factors affecting enzyme evolution can be obtained by examining nucleotide sequence variation in candidate genes such as Lactate dehydrogenase (Ldh). Two closely related Daphnia species can be distinguished by their electrophoretic Ldh genotype and habitat. Daphnia pulex populations are fixed for the S allele and inhabit temporary ponds, while D. pulicaria populations are fixed for the F allele and inhabit large stratified lakes. One locus is detected in most allozyme surveys, but genome sequencing has revealed two genes, LdhA and LdhB. Results: We sequenced both Ldh genes from 70 isolates of these two species from North America to determine if the association between Ldh genotype and habitat shows evidence for selection, and to elucidate the evolutionary history of the two genes. We found that alleles in the pond-dwelling D. pulex and in the lake-dwelling D. pulicaria form distinct groups at both loci, and the substitution of Glutamine (S) for Glutamic acid (F) at amino acid 229 likely causes the electrophoretic mobility shift in the LDHA protein. Nucleotide diversity in both Ldh genes is much lower in D. pulicaria than in D. pulex. Moreover, the lack of spatial structuring of the variation in both genes over a wide geographic area is consistent with a recent demographic expansion of lake populations. Neutrality tests indicate that both genes are under purifying selection, but the intensity is much stronger on LdhA. Conclusions: Although lake-dwelling D. pulicaria hybridizes with the other lineages in the pulex species complex, it remains distinct ecologically and genetically. This ecological divergence, coupled with the intensity of purifying selection on LdhA and the strong association between its genotype and habitat, suggests that experimental studies would be useful to determine if variation in molecular function provides evidence that LDHA variants are adaptive