ALBERT

Description: Nature Reviews Microbiology 13, 526 (2015). doi:10.1038/nrmicro3538 Author: Cláudio Nunes-Alves Persisters are dormant bacterial populations that contribute to multidrug tolerance (MDT) because they are able to escape treatment with most antibiotics, which target metabolically active cells. A new study now characterizes the molecular basis for persister generation involving HipA, a kinase that inhibits protein synthesis

Description: Nature Reviews Microbiology 13, 589 (2015). doi:10.1038/nrmicro3504 Authors: Peter Liehl, Vanessa Zuzarte-Luis & Maria M. Mota Many clinically relevant pathogens, including certain bacteria and protozoan parasites, have developed an intracellular lifestyle that enables them to nestle in customized vacuoles. Although these pathogens are protected from extracellular defences, recent findings indicate that host cells have evolved multiple strategies to unmask the pathogen

Description: Spliceosomal introns are a hallmark of eukaryotic genes that are hypothesized to play important roles in genome evolution but have poorly understood origins. Although most introns lack sequence homology to each other, new families of spliceosomal introns that are repeated hundreds of times in individual genomes have recently been discovered in a few organisms. The prevalence and conservation of these introner elements (IEs) or introner-like elements in other taxa, as well as their evolutionary relationships to regular spliceosomal introns, are still unknown. Here, we systematically investigate introns in the widespread marine green alga Micromonas and report new families of IEs, numerous intron presence–absence polymorphisms, and potential intron insertion hot-spots. The new families enabled identification of conserved IE secondary structure features and establishment of a novel general model for repetitive intron proliferation across genomes. Despite shared secondary structure, the IE families from each Micromonas lineage bear no obvious sequence similarity to those in the other lineages, suggesting that their appearance is intimately linked with the process of speciation. Two of the new IE families come from an Arctic culture ( Micromonas Clade E2) isolated from a polar region where abundance of this alga is increasing due to climate induced changes. The same two families were detected in metagenomic data from Antarctica—a system where Micromonas has never before been reported. Strikingly high identity between the Arctic isolate and Antarctic coding sequences that flank the IEs suggests connectivity between populations in the two polar systems that we postulate occurs through deep-sea currents. Recovery of Clade E2 sequences in North Atlantic Deep Waters beneath the Gulf Stream supports this hypothesis. Our research illuminates the dynamic relationships between an unusual class of repetitive introns, genome evolution, speciation, and global distribution of this sentinel marine alga.

Description: Cellular Ca 2+ homeostasis is tightly regulated and is pivotal to life. Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and ryanodine receptors (RyRs) are the major ion channels that regulate Ca 2+ release from intracellular stores. Although these channels have been extensively investigated in multicellular organisms, an appreciation of their evolution and the biology of orthologs in unicellular organisms is largely lacking. Extensive phylogenetic analyses reveal that the IP 3 R gene superfamily is ancient and diverged into two subfamilies, IP 3 R-A and IP 3 R-B/RyR, at the dawn of Opisthokonta. IP 3 R-B/RyR further diversified into IP 3 R-B and RyR at the stem of Filozoa. Subsequent evolution and speciation of Holozoa is associated with duplication of IP 3 R-A and RyR genes, and loss of IP 3 R-B in the vertebrate lineages. To gain insight into the properties of IP 3 R important for the challenges of multicellularity, the IP 3 R-A and IP 3 R-B family orthologs were cloned from Capsaspora owczarzaki, a close unicellular relative to Metazoa (designated as CO.IP 3 R-A and CO.IP 3 R-B). Both proteins were targeted to the endoplasmic reticulum. However, CO.IP 3 R-A, but strikingly not CO.IP 3 R-B, bound IP 3 , exhibited robust Ca 2+ release activity and associated with mammalian IP 3 Rs. These data indicate strongly that CO.IP 3 R-A as an exemplar of ancestral IP 3 R-A orthologs forms bona fide IP 3 -gated channels. Notably, however, CO.IP 3 R-A appears not to be regulated by Ca 2+ , ATP or Protein kinase A-phosphorylation. Collectively, our findings explore the origin, conservation, and diversification of IP 3 R gene families and provide insight into the functionality of ancestral IP 3 Rs and the added specialization of these proteins in Metazoa.

Description: Marriage rules, the community prescriptions that dictate who an individual can or cannot marry, are extremely diverse and universally present in traditional societies. A major focus of research in the early decades of modern anthropology, marriage rules impose social and economic forces that help structure societies and forge connections between them. However, in those early anthropological studies, the biological benefits or disadvantages of marriage rules could not be determined. We revisit this question by applying a novel simulation framework and genome-wide data to explore the effects of Asymmetric Prescriptive Alliance, an elaborate set of marriage rules that has been a focus of research for many anthropologists. Simulations show that strict adherence to these marriage rules reduces genetic diversity on the autosomes, X chromosome and mitochondrial DNA, but relaxed compliance produces genetic diversity similar to random mating. Genome-wide data from the Indonesian community of Rindi, one of the early study populations for Asymmetric Prescriptive Alliance, are more consistent with relaxed compliance than strict adherence. We therefore suggest that, in practice, marriage rules are treated with sufficient flexibility to allow social connectivity without significant degradation of biological diversity.

Description: The environment has profound effects on the expression of many traits and reaction norms describe the expression dynamics of a trait across a broad range of environmental conditions. Here, we analyze gene expression in Drosophila melanogaster across four different developmental temperatures (13–29 °C). Gene expression is highly plastic with 83.3% of the genes being differentially expressed. We distinguished three components of plasticity: 1) Dynamics of gene expression intensity (sum of change), 2) direction of change, and 3) curvature of the reaction norm (linear vs. quadratic). Studying their regulatory architecture we found that all three plasticity components were most strongly affected by the number of different transcription factors (TFs) binding to the target gene. More TFs were found in genes with less expression changes across temperatures. Although the effect of microRNAs was weaker, we consistently noted a trend in the opposite direction. The most plastic genes were regulated by fewer TFs and more microRNAs than less plastic genes. Different patterns of plasticity were also reflected by their functional characterization based on gene ontology. Our results suggest that reaction norms provide an important key to understand the functional requirements of natural populations exposed to variable environmental conditions.

Description: Recent advances in paleogenomic technologies have enabled an increasingly detailed understanding of the evolutionary relationships of now-extinct mammalian taxa. However, a number of enigmatic Quaternary species have never been characterized with molecular data, often because available fossils are rare or are found in environments that are not optimal for DNA preservation. Here, we analyze paleogenomic data extracted from bones attributed to the late Pleistocene western camel, Camelops cf. hesternus, a species that was distributed across central and western North America until its extinction approximately 13,000 years ago. Despite a modal sequence length of only around 35 base pairs, we reconstructed high-coverage complete mitochondrial genomes and low-coverage partial nuclear genomes for each specimen. We find that Camelops is sister to African and Asian bactrian and dromedary camels, to the exclusion of South American camelids (llamas, guanacos, alpacas, and vicuñas). These results contradict previous morphology-based phylogenetic models for Camelops , which suggest instead a closer relationship between Camelops and the South American camelids. The molecular data imply a Late Miocene divergence of the Camelops clade from lineages that separately gave rise to the extant camels of Eurasia. Our results demonstrate the increasing capacity of modern paleogenomic methods to resolve evolutionary relationships among distantly related lineages.

Description: Recent developments in the analysis of amino acid covariation are leading to breakthroughs in protein structure prediction, protein design, and prediction of the interactome. It is assumed that observed patterns of covariation are caused by molecular coevolution, where substitutions at one site affect the evolutionary forces acting at neighboring sites. Our theoretical and empirical results cast doubt on this assumption. We demonstrate that the strongest coevolutionary signal is a decrease in evolutionary rate and that unfeasibly long times are required to produce coordinated substitutions. We find that covarying substitutions are mostly found on different branches of the phylogenetic tree, indicating that they are independent events that may or may not be attributable to coevolution. These observations undermine the hypothesis that molecular coevolution is the primary cause of the covariation signal. In contrast, we find that the pairs of residues with the strongest covariation signal tend to have low evolutionary rates, and that it is this low rate that gives rise to the covariation signal. Slowly evolving residue pairs are disproportionately located in the protein’s core, which explains covariation methods’ ability to detect pairs of residues that are close in three dimensions. These observations lead us to propose the "coevolution paradox": The strength of coevolution required to cause coordinated changes means the evolutionary rate is so low that such changes are highly unlikely to occur. As modern covariation methods may lead to breakthroughs in structural genomics, it is critical to recognize their biases and limitations.

Description: We present a modified GPU (graphics processing unit) version of MrBayes, called ta(MC) 3 (GPU MrBayes V3.1), for Bayesian phylogenetic inference on protein data sets. Our main contributions are 1) utilizing 64-bit variables, thereby enabling ta(MC) 3 to process larger data sets than MrBayes; and 2) to use Kahan summation to improve accuracy, convergence rates, and consequently runtime. Versus the current fastest software, we achieve a speedup of up to around 2.5 (and up to around 90 vs. serial MrBayes), and more on multi-GPU hardware. GPU MrBayes V3.1 is available from http://sourceforge.net/projects/mrbayes-gpu/ .

Description: Understanding the genetic and molecular bases of the ability to distinguish self from nonself (allorecognition) and mechanisms underlying evolution of allorecognition systems is an important endeavor for understanding cases where it becomes dysfunctional, such as in autoimmune disorders. In filamentous fungi, allorecognition can result in vegetative or heterokaryon incompatibility, which is a type of programmed cell death that occurs following fusion of genetically different cells. Allorecognition is genetically controlled by het loci, with coexpression of any combination of incompatible alleles triggering vegetative incompatibility. Herein, we identified, characterized, and inferred the evolutionary history of candidate het loci in the filamentous fungus Neurospora crassa . As characterized het loci encode proteins carrying an HET domain, we annotated HET domain genes in 25 isolates from a natural population along with the N. crassa reference genome using resequencing data. Because allorecognition systems can be affected by frequency-dependent selection favoring rare alleles (i.e., balancing selection), we mined resequencing data for HET domain loci whose alleles displayed elevated levels of variability, excess of intermediate frequency alleles, and deep gene genealogies. From these analyses, 34 HET domain loci were identified as likely to be under balancing selection. Using transformation, incompatibility assays and genetic analyses, we determined that one of these candidates functioned as a het locus ( het-e ). The het-e locus has three divergent allelic groups that showed signatures of positive selection, intra- and intergroup recombination, and trans-species polymorphism. Our findings represent a compelling case of balancing selection functioning on multiple alleles across multiple loci potentially involved in allorecognition.

Description: Scoring the impact of noncoding variation on the function of cis -regulatory regions, on their chromatin state, and on the qualitative and quantitative expression levels of target genes is a fundamental problem in evolutionary genomics. A particular challenge is how to model the divergence of quantitative traits and to identify relationships between the changes across the different levels of the genome, the chromatin activity landscape, and the transcriptome. Here, we examine the use of the Ornstein–Uhlenbeck (OU) model to infer selection at the level of predicted cis -regulatory modules (CRMs), and link these with changes in transcription factor binding and chromatin activity. Using publicly available cross-species ChIP-Seq and STARR-Seq data we show how OU can be applied genome-wide to identify candidate transcription factors for which binding site and CRM turnover is correlated with changes in regulatory activity. Next, we profile open chromatin in the developing eye across three Drosophila species. We identify the recognition motifs of the chromatin remodelers, Trithorax-like and Grainyhead as mostly correlating with species-specific changes in open chromatin. In conclusion, we show in this study that CRM scores can be used as quantitative traits and that motif discovery approaches can be extended towards more complex models of divergence.

Description: How populations that inhabit the same geographical area become genetically differentiated is not clear. To investigate this, we characterized phenotypic and genetic differences between two populations of Saccharomyces cerevisiae that in some cases inhabit the same environment but show relatively little gene flow. We profiled stress sensitivity in a group of vineyard isolates and a group of oak-soil strains and found several niche-related phenotypes that distinguish the populations. We performed bulk-segregant mapping on two of the distinguishing traits: The vineyard-specific ability to grow in grape juice and oak-specific tolerance to the cell wall damaging drug Congo red. To implicate causal genes, we also performed a chemical genomic screen in the lab-strain deletion collection and identified many important genes that fell under quantitative trait loci peaks. One gene important for growth in grape juice and identified by both the mapping and the screen was SSU1 , a sulfite-nitrite pump implicated in wine fermentations. The beneficial allele is generated by a known translocation that we reasoned may also serve as a genetic barrier. We found that the translocation is prevalent in vineyard strains, but absent in oak strains, and presents a postzygotic barrier to spore viability. Furthermore, the translocation was associated with a fitness cost to the rapid growth rate seen in oak-soil strains. Our results reveal the translocation as a dual-function locus that enforces ecological differentiation while producing a genetic barrier to gene flow in these sympatric populations.

Description: Species tree reconstruction has been a subject of substantial research due to its central role across biology and medicine. A species tree is often reconstructed using a set of gene trees or by directly using sequence data. In either of these cases, one of the main confounding phenomena is the discordance between a species tree and a gene tree due to evolutionary events such as duplications and losses. Probabilistic methods can resolve the discordance by coestimating gene trees and the species tree but this approach poses a scalability problem for larger data sets. We present MixTreEM-DLRS: A two-phase approach for reconstructing a species tree in the presence of gene duplications and losses. In the first phase, MixTreEM, a novel structural expectation maximization algorithm based on a mixture model is used to reconstruct a set of candidate species trees, given sequence data for monocopy gene families from the genomes under study. In the second phase, PrIME-DLRS, a method based on the DLRS model (Åkerborg O, Sennblad B, Arvestad L, Lagergren J. 2009. Simultaneous Bayesian gene tree reconstruction and reconciliation analysis. Proc Natl Acad Sci U S A. 106(14):5714–5719), is used for selecting the best species tree. PrIME-DLRS can handle multicopy gene families since DLRS, apart from modeling sequence evolution, models gene duplication and loss using a gene evolution model (Arvestad L, Lagergren J, Sennblad B. 2009. The gene evolution model and computing its associated probabilities. J ACM. 56(2):1–44). We evaluate MixTreEM-DLRS using synthetic and biological data, and compare its performance with a recent genome-scale species tree reconstruction method PHYLDOG ( Boussau B, Szöllősi GJ, Duret L, Gouy M, Tannier E, Daubin V. 2013 . Genome-scale coestimation of species and gene trees. Genome Res. 23(2):323–330) as well as with a fast parsimony-based algorithm Duptree (Wehe A, Bansal MS, Burleigh JG, Eulenstein O. 2008. Duptree: a program for large-scale phylogenetic analyses using gene tree parsimony. Bioinformatics 24(13):1540–1541). Our method is competitive with PHYLDOG in terms of accuracy and runs significantly faster and our method outperforms Duptree in accuracy. The analysis constituted by MixTreEM without DLRS may also be used for selecting the target species tree, yielding a fast and yet accurate algorithm for larger data sets. MixTreEM is freely available at http://prime.scilifelab.se/mixtreem/ .

Description: Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X–Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (~5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X–Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.

Description: Environmental control of flowering allows plant reproduction to occur under optimal conditions and facilitates adaptation to different locations. At high latitude, flowering of many plants is controlled by seasonal changes in day length. The photoperiodic flowering pathway confers this response in the Brassicaceae, which colonized temperate latitudes after divergence from the Cleomaceae, their subtropical sister family. The CONSTANS (CO) transcription factor of Arabidopsis thaliana , a member of the Brassicaceae, is central to the photoperiodic flowering response and shows characteristic patterns of transcription required for day-length sensing. CO is believed to be widely conserved among flowering plants; however, we show that it arose after gene duplication at the root of the Brassicaceae followed by divergence of transcriptional regulation and protein function. CO has two close homologs, CONSTANS-LIKE1 ( COL1 ) and COL2 , which are related to CO by tandem duplication and whole-genome duplication, respectively. The single CO homolog present in the Cleomaceae shows transcriptional and functional features similar to those of COL1 and COL2 , suggesting that these were ancestral. We detect cis -regulatory and codon changes characteristic of CO and use transgenic assays to demonstrate their significance in the day-length-dependent activation of the CO target gene FLOWERING LOCUS T. Thus, the function of CO as a potent photoperiodic flowering switch evolved in the Brassicaceae after gene duplication. The origin of CO may have contributed to the range expansion of the Brassicaceae and suggests that in other families CO genes involved in photoperiodic flowering arose by convergent evolution.

Description: The high regulatory complexity of vertebrates has been related to two rounds of whole genome duplication (2R-WGD) that occurred before the divergence of the major vertebrate groups. Following these events, many developmental transcription factors (TFs) were retained in multiple copies and subsequently specialized in diverse functions, whereas others reverted to their singleton state. TFs are known to be generally rich in amino acid repeats or low-complexity regions (LCRs), such as polyalanine or polyglutamine runs, which can evolve rapidly and potentially influence the transcriptional activity of the protein. Here we test the hypothesis that LCRs have played a major role in the diversification of TF gene duplicates. We find that nearly half of the TF gene families originated during the 2R-WGD contains LCRs. The number of gene duplicates with LCRs is 155 out of 550 analyzed (28%), about twice as many as the number of single copy genes with LCRs (15 out of 115, 13%). In addition, duplicated TFs preferentially accumulate certain LCR types, the most prominent of which are alanine repeats. We experimentally test the role of alanine-rich LCRs in two different TF gene families, PHOX2A/PHOX2B and LHX2/LHX9. In both cases, the presence of the alanine-rich LCR in one of the copies (PHOX2B and LHX2) significantly increases the capacity of the TF to activate transcription. Taken together, the results provide strong evidence that LCRs are important driving forces of evolutionary change in duplicated genes.

Description: Studies of species with continental distributions continue to identify intraspecific lineages despite continuous habitat. Lineages may form due to isolation by distance, adaptation, divergence across barriers, or genetic drift following range expansion. We investigated lineage diversification and admixture within American black bears ( Ursus americanus ) across their range using 22 k single nucleotide polymorphisms and mitochondrial DNA sequences. We identified three subcontinental nuclear clusters which we further divided into nine geographic regions: Alaskan (Alaska-East), eastern (Central Interior Highlands, Great Lakes, Northeast, Southeast), and western (Alaska-West, West, Pacific Coast, Southwest). We estimated that the western cluster diverged 67 ka, before eastern and Alaskan divergence 31 ka; these divergence dates contrasted with those from the mitochondrial genome where clades A and B diverged 1.07 Ma, and clades A-east and A-west diverged 169 ka. We combined estimates of divergence timing with hindcast species distribution models to infer glacial refugia for the species in Beringia, Pacific Northwest, Southwest, and Southeast. Our results show a complex arrangement of admixture due to expansion out of multiple refugia. The delineation of the genomic population clusters was inconsistent with the ranges for 16 previously described subspecies. Ranges for U. a. pugnax and U. a. cinnamomum were concordant with admixed clusters, calling into question how to order taxa below the species level. Additionally, our finding that U. a. floridanus has not diverged from U. a. americanus also suggests that morphology and genetics should be reanalyzed to assess taxonomic designations relevant to the conservation management of the species.

Description: Deinococcus bacteria are extremely resistant to radiation, oxidation, and desiccation. Resilience to these factors has been suggested to be due to enhanced damage prevention and repair mechanisms, as well as highly efficient antioxidant protection systems. Here, using mutation-accumulation experiments, we find that the GC-rich Deinococcus radiodurans has an overall background genomic mutation rate similar to that of E. coli , but differs in mutation spectrum, with the A/T to G/C mutation rate (based on a total count of 88 A:T-〉G:C transitions and 82 A:T-〉C:G transversions) per site per generation higher than that in the other direction (based on a total count of 157 G:C-〉A:T transitions and 33 G:C-〉T:A transversions). We propose that this unique spectrum is shaped mainly by the abundant uracil DNA glycosylases reducing G:C-〉A:T transitions, adenine methylation elevating A:T-〉C:G transversions, and absence of cytosine methylation decreasing G:C-〉A:T transitions. As opposed to the greater than 100 x elevation of the mutation rate in MMR – (DNA Mismatch Repair deficient) strains of most other organisms, MMR – D. radiodurans only exhibits a 4-fold elevation, raising the possibility that other DNA repair mechanisms compensate for a relatively low-efficiency DNA MMR pathway. As D. radiodurans has plentiful insertion sequence (IS) elements in the genome and the activities of IS elements are rarely directly explored, we also estimated the insertion (transposition) rate of the IS elements to be 2.50 x 10 –3 per genome per generation in the wild-type strain; knocking out MMR did not elevate the IS element insertion rate in this organism.

Description: Because mating systems affect population genetics and ecology, they are expected to impact the molecular evolution of species. Self-fertilizing species experience reduced effective population size, recombination rates, and heterozygosity, which in turn should decrease the efficacy of natural selection, both adaptive and purifying, and the strength of meiotic drive processes such as GC-biased gene conversion. The empirical evidence is only partly congruent with these predictions, depending on the analyzed species, some, but not all, of the expected effects have been observed. One possible reason is that self-fertilization is an evolutionary dead-end, so that most current selfers recently evolved self-fertilization, and their genome has not yet been strongly impacted by selfing. Here, we investigate the molecular evolution of two groups of freshwater snails in which mating systems have likely been stable for several millions of years. Analyzing coding sequence polymorphism, divergence, and expression levels, we report a strongly reduced genetic diversity, decreased efficacy of purifying selection, slower rate of adaptive evolution, and weakened codon usage bias/GC-biased gene conversion in the selfer Galba compared with the outcrosser Physa , in full agreement with theoretical expectations. Our results demonstrate that self-fertilization, when effective in the long run, is a major driver of population genomic and molecular evolutionary processes. Despite the genomic effects of selfing, Galba truncatula seems to escape the demographic consequences of the genetic load. We suggest that the particular ecology of the species may buffer the negative consequences of selfing, shedding new light on the dead-end hypothesis.

Description: Nature Reviews Microbiology 13, 459 (2015). doi:10.1038/nrmicro3547 Author: Naomi Attar In type II CRISPR−Cas adaptive immunity systems, a CRISPR RNA (crRNA) forms a hybrid structure with a trans-activating crRNA (tracrRNA) that, together with recognition of a protospacer adjacent motif (PAM), guides the Cas9 endonuclease protein to foreign DNA. Jiang et al. solved a

Description: Nature Reviews Microbiology 13, 573 (2015). doi:10.1038/nrmicro3519 Authors: Gabrielle A. Josling & Manuel Llinás Malaria is a devastating infectious disease that is caused by blood-borne apicomplexan parasites of the genus Plasmodium. These pathogens have a complex lifecycle, which includes development in the anopheline mosquito vector and in the liver and red blood cells of mammalian hosts, a process

Description: Nature Reviews Microbiology 13, 559 (2015). doi:10.1038/nrmicro3508 Authors: Athanasios Typas & Victor Sourjik Distinct cellular functions are executed by separate groups of proteins, organized into complexes or functional modules, which are ultimately interconnected in cell-wide protein networks. Understanding the structures and operational modes of these networks is one of the next great challenges in biology, and microorganisms are

Description: Nature Reviews Microbiology 13, 527 (2015). doi:10.1038/nrmicro3544 Author: Naomi Attar Most metagenomic analysis methods are based on the relative abundance of a marker gene or of sets of genes in functional pathways. However, these methods offer a static view of microbial composition and provide no information on growth dynamics. Korem et al. devised a

Description: Nature Reviews Microbiology 13, 544 (2015). doi:10.1038/nrmicro3506 Authors: You Li, Daisuke Yamane, Takahiro Masaki & Stanley M. Lemon Hepatitis C virus (HCV) is an unusual RNA virus that has a striking capacity to persist for the remaining life of the host in the majority of infected individuals. In order to persist, HCV must balance viral RNA synthesis and decay in infected cells. In

Description: Nature Reviews Microbiology 13, 459 (2015). doi:10.1038/nrmicro3536 Author: Naomi Attar The 'MinION' nanopore sequencer works by applying an ionic current across an electrically resistant membrane permeabilized by protein nanopores. The sequence of DNA molecules passing through each nanopore is determined by measuring changes to this current. Nanopore sequencers can vastly increase the permissible length of

Description: Nature Reviews Microbiology 13, 526 (2015). doi:10.1038/nrmicro3539 Author: Cláudio Nunes-Alves Several bacterial pathogens, including Vibrio cholerae, use toxins containing actin crosslinking domains (ACDs) to interfere with the ability of the host cell to polymerize actin. The toxicity of ACD-containing toxins is thought to arise via sequestration of bulk amounts of actin as non-functional oligomers,

Description: Nature Reviews Microbiology 13, 527 (2015). doi:10.1038/nrmicro3543 Author: Naomi Attar The bacterial type VI secretion system (T6SS) is a nanomachine that punctures the membranes of competing bacteria and, during pathogenesis, those of host cells. In addition to the tail, which is composed of a contractile sheath and a tipped arrow that punctures the membrane of

Description: Nature Reviews Microbiology 13, 525 (2015). doi:10.1038/nrmicro3542 Author: Sarah Crunkhorn Two recent studies report the development of monoclonal antibodies directed against novel epitopes within the viral envelope protein of dengue virus that prevent severe infection in mouse models.

Description: Nature Reviews Microbiology 13, 527 (2015). doi:10.1038/nrmicro3545 Author: Naomi Attar Hypervirulent Klebsiella pneumoniae is a globally emerging pathogen that causes pyogenic liver abscesses. Hypervirulent strains are restricted to capsular serotypes K1 and K2 and are particularly associated with the clonal complex K1 CC23. To understand the evolution of hypervirulence, Struve et al. sequenced

Description: Nature Reviews Microbiology 13, 529 (2015). doi:10.1038/nrmicro3521 Authors: Vilasack Thammavongsa, Hwan Keun Kim, Dominique Missiakas & Olaf Schneewind Staphylococcus aureus, a bacterial commensal of the human nares and skin, is a frequent cause of soft tissue and bloodstream infections. A hallmark of staphylococcal infections is their frequent recurrence, even when treated with antibiotics and surgical intervention, which demonstrates the bacterium's ability to

Description: Nature Reviews Microbiology 13, 526 (2015). doi:10.1038/nrmicro3541 Author: Denise Waldron This study uses a new tool, VirSorter, to search for virus genomes within all publicly available bacterial and archaeal genomic datasets and finds 264 new candidate viral genera.

Description: Nature Reviews Microbiology 13, 526 (2015). doi:10.1038/nrmicro3540 Author: Cláudio Nunes-Alves The Ebola epidemic in West Africa is still ongoing, but a new study reports that a ring vaccination strategy — in which contacts, and contacts of contacts, of an infected patient are vaccinated — using rVSV-ZEBOV was successful in preventing infection. The vaccine consists of

Description: Nature Reviews Microbiology 13, 600 (2015). doi:10.1038/nrmicro3561 Author: Naomi Attar 80% of land plants have symbiotic arbuscular mycorrhizal fungi, which are named after the 'arbuscule' structures that are used for nutrient exchange. Despite their ecological importance, surveys of these fungi have been limited, as these taxa are mostly unculturable, but have pointed to an endemic

Description: Nature Reviews Microbiology 13, 600 (2015). doi:10.1038/nrmicro3554 Author: Naomi Attar A new study shows that variability of contact-dependent growth inhibition toxins is linked to a large repertoire of cell-entry pathways.

Description: Nature Reviews Microbiology 13, 600 (2015). doi:10.1038/nrmicro3550 Author: Naomi Attar A new study delineates the origin and transmission pattern of Lassa virus, an important human pathogen.

Description: Nature Reviews Microbiology 13, 599 (2015). doi:10.1038/nrmicro3553 Author: Cláudio Nunes-Alves These two studies report novel vaccination strategies to elicit antibodies that target the conserved stem region of haemagglutinin in order to generate protective immune responses against multiple influenza subtypes.

Description: Nature Reviews Microbiology 13, 600 (2015). doi:10.1038/nrmicro3560 Author: Naomi Attar Type VI secretion systems (T6SSs) and their effectors can function in both pathogenesis and interbacterial competition. Vibrio alginolyticus, a marine pathogen that can infect humans, encodes two T6SSs, but only a single antibacterial effector, Va02265, had been described for these systems. Of note, Va02265

Description: Nature Reviews Microbiology 13, 601 (2015). doi:10.1038/nrmicro3559 Author: Cláudio Nunes-Alves Following infection, Legionella pneumophila is able to hijack several cellular processes in order to establish the Legionella-containing vacuole (LCV), which enables bacterial replication within the host cell. However, how the bacteria acquire essential nutrients, such as iron, within the LCV is unknown. Isaac

Description: Nature Reviews Microbiology 13, 602 (2015). doi:10.1038/nrmicro3556 Author: Denise Waldron A new study reports that the genetic diversification of Pseudomonas aeruginosa within the lungs of patients with cystic fibrosis (CF) is driven by regional isolation.

Description: Nature Reviews Microbiology 13, 601 (2015). doi:10.1038/nrmicro3558 Author: Cláudio Nunes-Alves The study of hepatitis C virus (HCV) has been hampered by the inability to grow viral clinical isolates in cell culture. Saeed et al. postulated that this was due to the lack of essential factors in cell lines, and transduced human hepatoma cells with

Description: Nature Reviews Microbiology 13, 600 (2015). doi:10.1038/nrmicro3562 Author: Naomi Attar Intestinal commensals inhabit an anaerobic environment where conventional fluorescent protein tags are not functional. Now, Kasper and colleagues use bacterial biosynthesis and click chemistry to fluorescently label the polysaccharides that are shed from the capsules of commensal bacteria. As a first step, cultures are grown

Description: Nature Reviews Microbiology 13, 603 (2015). doi:10.1038/nrmicro3551 Authors: Christian K. Owusu & Hayley M. Bennett This month's Genome Watch explores apicomplexan adaptation to parasitism.

Description: Nature Reviews Microbiology 13, 605 (2015). doi:10.1038/nrmicro3525 Authors: Carmen Schwechheimer & Meta J. Kuehn Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling

Description: Nature Reviews Microbiology 13, 601 (2015). doi:10.1038/nrmicro3557 Author: Cláudio Nunes-Alves To understand the bacterial–fungal interactions between Pseudomonas aeruginosa and Candida albicans, Lopez-Medina et al. used a mouse model of microbial gastrointestinal colonization and dissemination. They found that co-colonization with C. albicans inhibited the virulence of P. aeruginosa, although the fungus

Description: Nature Reviews Microbiology 13, 651 (2015). doi:10.1038/nrmicro3523 Author: Carl Nathan As foundations and governments mobilize to tackle antimicrobial resistance (AMR), several experiments in academic–industrial collaboration have emerged. Here, I examine two historical precedents, the Penicillin Project and the Malaria Project of the Second World War, and two contemporary examples, the Tuberculosis Drug Accelerator programme and

Description: Nature Reviews Microbiology 13, 602 (2015). doi:10.1038/nrmicro3555 Author: Elisabeth Kugelberg Malaria infection modifies endemic Burkitt lymphomas by inducing a prolonged expansion of germinal centre B cell numbers.

Description: Nature Reviews Microbiology 13, 631 (2015). doi:10.1038/nrmicro3520 Authors: Craig R. Roy & Jacqueline Cherfils Fic proteins are a family of proteins characterized by the presence of a conserved FIC domain that is involved in the modification of protein substrates by the addition of phosphate-containing compounds, including AMP and other nucleoside monophosphates, phosphocholine and phosphate. Fic proteins are widespread in

Description: Nature Reviews Microbiology 13, 641 (2015). doi:10.1038/nrmicro3527 Authors: Ron Feiner, Tal Argov, Lev Rabinovich, Nadejda Sigal, Ilya Borovok & Anat A. Herskovits Unlike lytic phages, temperate phages that enter lysogeny maintain a long-term association with their bacterial host. In this context, mutually beneficial interactions can evolve that support efficient reproduction of both phages and bacteria. Temperate phages are integrated into the bacterial chromosome as large DNA insertions

Description: Nature Reviews Microbiology 13, 620 (2015). doi:10.1038/nrmicro3480 Authors: Lisa Brown, Julie M. Wolf, Rafael Prados-Rosales & Arturo Casadevall Extracellular vesicles (EVs) are produced by all domains of life. In Gram-negative bacteria, EVs are produced by the pinching off of the outer membrane; however, how EVs escape the thick cell walls of Gram-positive bacteria, mycobacteria and fungi is still unknown. Nonetheless, EVs have been

Description: Nucleobase modifications dramatically alter nucleic acid structure and thermodynamics. 2-thiouridine (s 2 U) is a modified nucleobase found in tRNAs and known to stabilize U:A base pairs and destabilize U:G wobble pairs. The recently reported crystal structures of s 2 U-containing RNA duplexes do not entirely explain the mechanisms responsible for the stabilizing effect of s 2 U or whether this effect is entropic or enthalpic in origin. We present here thermodynamic evaluations of duplex formation using ITC and UV thermal denaturation with RNA duplexes containing internal s 2 U:A and s 2 U:U pairs and their native counterparts. These results indicate that s 2 U stabilizes both duplexes. The stabilizing effect is entropic in origin and likely results from the s 2 U-induced preorganization of the single-stranded RNA prior to hybridization. The same preorganizing effect is likely responsible for structurally resolving the s 2 U:U pair-containing duplex into a single conformation with a well-defined H-bond geometry. We also evaluate the effect of s 2 U on single strand conformation using UV- and CD-monitored thermal denaturation and on nucleoside conformation using 1 H NMR spectroscopy, MD and umbrella sampling. These results provide insights into the effects that nucleobase modification has on RNA structure and thermodynamics and inform efforts toward improving both ribozyme-catalyzed and nonenzymatic RNA copying.

Description: DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl 2 , NiCl 2 , ZnCl 2 and MnCl 2 . In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion.

Description: The sequencing of the full transcriptome (RNA-seq) has become the preferred choice for the measurement of genome-wide gene expression. Despite its widespread use, challenges remain in RNA-seq data analysis. One often-overlooked aspect is normalization. Despite the fact that a variety of factors or ‘batch effects’ can contribute unwanted variation to the data, commonly used RNA-seq normalization methods only correct for sequencing depth. The study of gene expression is particularly problematic when it is influenced simultaneously by a variety of biological factors in addition to the one of interest. Using examples from experimental neuroscience, we show that batch effects can dominate the signal of interest; and that the choice of normalization method affects the power and reproducibility of the results. While commonly used global normalization methods are not able to adequately normalize the data, more recently developed RNA-seq normalization can. We focus on one particular method, RUVSeq and show that it is able to increase power and biological insight of the results. Finally, we provide a tutorial outlining the implementation of RUVSeq normalization that is applicable to a broad range of studies as well as meta-analysis of publicly available data.

Description: Many antiproliferative G-quadruplexes (G4s) arise from the folding of GT-rich strands. Among these, the Thrombin Binding Aptamer (TBA), as a rare example, adopts a monomolecular well-defined G4 structure. Nevertheless, the potential anticancer properties of TBA are severely hampered by its anticoagulant action and, consequently, no related studies have appeared so far in the literature. We wish to report here that suitable chemical modifications in the TBA sequence can preserve its antiproliferative over anticoagulant activity. Particularly, we replaced one residue of the TT or TGT loops with a dibenzyl linker to develop seven new quadruplex-forming TBA based sequences (TBA-bs), which were studied for their structural (CD, CD melting, 1D NMR) and biological (fibrinogen, PT and MTT assays) properties. The three-dimensional structures of the TBA-bs modified at T13 (TBA-bs13) or T12 (TBA-bs12), the former endowed with selective antiproliferative activity, and the latter acting as potently as TBA in both coagulation and MTT assays, were further studied by 2D NMR restrained molecular mechanics. The comparative structural analyses indicated that neither the stability, nor the topology of the G4s, but the different localization of the two benzene rings of the linker was responsible for the loss of the antithrombin activity for TBA-bs13.

Description: A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.

Description: Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.

Description: Sequence alignment is a long standing problem in bioinformatics. The Basic Local Alignment Search Tool (BLAST) is one of the most popular and fundamental alignment tools. The explosive growth of biological sequences calls for speedup of sequence alignment tools such as BLAST. To this end, we develop high speed BLASTN (HS-BLASTN), a parallel and fast nucleotide database search tool that accelerates MegaBLAST—the default module of NCBI-BLASTN. HS-BLASTN builds a new lookup table using the FMD-index of the database and employs an accurate and effective seeding method to find short stretches of identities (called seeds) between the query and the database. HS-BLASTN produces the same alignment results as MegaBLAST and its computational speed is much faster than MegaBLAST. Specifically, our experiments conducted on a 12-core server show that HS-BLASTN can be 22 times faster than MegaBLAST and exhibits better parallel performance than MegaBLAST. HS-BLASTN is written in C++ and the related source code is available at https://github.com/chenying2016/queries under the GPLv3 license.

Description: Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo . Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors.

Description: Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3' untranslated region (3' UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.

Description: The emergence of multidrug-resistant pathogens will make current antibiotics ineffective. For linezolid, a member of the novel oxazolidinone class of antibiotics, 10 nucleotide mutations in the ribosome have been described conferring resistance. Hypotheses for how these mutations affect antibiotics binding have been derived based on comparative crystallographic studies. However, a detailed description at the atomistic level of how remote mutations exert long-distance effects has remained elusive. Here, we show that the G2032A-C2499A double mutation, located 〉 10 Å away from the antibiotic, confers linezolid resistance by a complex set of effects that percolate to the binding site. By molecular dynamics simulations and free energy calculations, we identify U2504 and C2452 as spearheads among binding site nucleotides that exert the most immediate effect on linezolid binding. Structural reorganizations within the ribosomal subunit due to the mutations are likely associated with mutually compensating changes in the effective energy. Furthermore, we suggest two main routes of information transfer from the mutation sites to U2504 and C2452. Between these, we observe cross-talk, which suggests that synergistic effects observed for the two mutations arise in an indirect manner. These results should be relevant for the development of oxazolidinone derivatives that are active against linezolid-resistant strains.

Description: Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1 , or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

Description: Clonal populations accumulate mutations over time, resulting in different haplotypes. Deep sequencing of such a population in principle provides information to reconstruct these haplotypes and the frequency at which the haplotypes occur. However, this reconstruction is technically not trivial, especially not in clonal systems with a relatively low mutation frequency. The low number of segregating sites in those systems adds ambiguity to the haplotype phasing and thus obviates the reconstruction of genome-wide haplotypes based on sequence overlap information. Therefore, we present EVORhA, a haplotype reconstruction method that complements phasing information in the non-empty read overlap with the frequency estimations of inferred local haplotypes. As was shown with simulated data, as soon as read lengths and/or mutation rates become restrictive for state-of-the-art methods, the use of this additional frequency information allows EVORhA to still reliably reconstruct genome-wide haplotypes. On real data, we show the applicability of the method in reconstructing the population composition of evolved bacterial populations and in decomposing mixed bacterial infections from clinical samples.

Description: Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Description: Recent releases of genome three-dimensional (3D) structures have the potential to transform our understanding of genomes. Nonetheless, the storage technology and visualization tools need to evolve to offer to the scientific community fast and convenient access to these data. We introduce simultaneously a database system to store and query 3D genomic data ( 3DBG ), and a 3D genome browser to visualize and explore 3D genome structures ( 3DGB ). We benchmark 3DBG against state-of-the-art systems and demonstrate that it is faster than previous solutions, and importantly gracefully scales with the size of data. We also illustrate the usefulness of our 3D genome Web browser to explore human genome structures. The 3D genome browser is available at http://3dgb.cs.mcgill.ca/ .

Description: Due to their relatively low-cost per sample and broad, gene-centric coverage of CpGs across the human genome, Illumina's 450k arrays are widely used in large scale differential methylation studies. However, by their very nature, large studies are particularly susceptible to the effects of unwanted variation. The effects of unwanted variation have been extensively documented in gene expression array studies and numerous methods have been developed to mitigate these effects. However, there has been much less research focused on the appropriate methodology to use for accounting for unwanted variation in methylation array studies. Here we present a novel 2-stage approach using RUV-inverse in a differential methylation analysis of 450k data and show that it outperforms existing methods.

Description: Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β– variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response.

Description: The selfing syndrome constitutes a suite of floral and reproductive trait changes that have evolved repeatedly across many evolutionary lineages in response to the shift to selfing. Convergent evolution of the selfing syndrome suggests that these changes are adaptive, yet our understanding of the detailed molecular genetic basis of the selfing syndrome remains limited. Here, we investigate the role of cis- regulatory changes during the recent evolution of the selfing syndrome in Capsella rubella , which split from the outcrosser Capsella grandiflora less than 200 ka. We assess allele-specific expression (ASE) in leaves and flower buds at a total of 18,452 genes in three interspecific F1 C. grandiflora x C. rubella hybrids. Using a hierarchical Bayesian approach that accounts for technical variation using genomic reads, we find evidence for extensive cis- regulatory changes. On average, 44% of the assayed genes show evidence of ASE; however, only 6% show strong allelic expression biases. Flower buds, but not leaves, show an enrichment of cis- regulatory changes in genomic regions responsible for floral and reproductive trait divergence between C. rubella and C. grandiflora . We further detected an excess of heterozygous transposable element (TE) insertions near genes with ASE, and TE insertions targeted by uniquely mapping 24-nt small RNAs were associated with reduced expression of nearby genes. Our results suggest that cis -regulatory changes have been important during the recent adaptive floral evolution in Capsella and that differences in TE dynamics between selfing and outcrossing species could be important for rapid regulatory divergence in association with mating system shifts.

Description: The phenomenon of male sterility in interspecies hybrids has been observed for over a century, however, few genes influencing this recurrent phenotype have been identified. Genetic investigations have been primarily limited to a small number of model organisms, thus limiting our understanding of the underlying molecular basis of this well-documented "rule of speciation." We utilized two interspecies hybrid cat breeds in a genome-wide association study employing the Illumina 63 K single-nucleotide polymorphism array. Collectively, we identified eight autosomal genes/gene regions underlying associations with hybrid male sterility (HMS) involved in the function of the blood-testis barrier, gamete structural development, and transcriptional regulation. We also identified several candidate hybrid sterility regions on the X chromosome, with most residing in close proximity to complex duplicated regions. Differential gene expression analyses revealed significant chromosome-wide upregulation of X chromosome transcripts in testes of sterile hybrids, which were enriched for genes involved in chromatin regulation of gene expression. Our expression results parallel those reported in Mus hybrids, supporting the "Large X-Effect" in mammalian HMS and the potential epigenetic basis for this phenomenon. These results support the value of the interspecies feline model as a powerful tool for comparison to rodent models of HMS, demonstrating unique aspects and potential commonalities that underpin mammalian reproductive isolation.

Description: Horizontal gene transfer threatens the therapeutic success of antibiotics by facilitating the rapid dissemination of resistance alleles among bacterial species. The conjugative mobile element Tn916 provides an excellent context for examining the role of adaptive parasexuality as it carries the tetracycline-resistance allele tetM and has been identified in a wide range of pathogens. We have used a combination of experimental evolution and allelic frequency measurements to gain insights into the adaptive trajectories leading to tigecycline resistance in a hospital strain of Enterococcus faecalis and predict what mechanisms of resistance are most likely to appear in the clinical setting. Here, we show that antibiotic selection led to the near fixation of adaptive alleles that simultaneously altered TetM expression and produced remarkably increased levels of Tn916 horizontal gene transfer. In the absence of drug, approximately 1 in 120,000 of the nonadapted E. faecalis S613 cells had an excised copy of Tn916, whereas nearly 1 in 50 cells had an excised copy of Tn916 upon selection for resistance resulting in a more than 1,000-fold increase in conjugation rates. We also show that tigecycline, a translation inhibitor, selected for a mutation in the ribosomal S10 protein. Our results show the first example of mutations that concurrently confer resistance to an antibiotic and lead to constitutive conjugal-transfer of the resistance allele. Selection created a highly parasexual phenotype and high frequency of Tn916 jumping demonstrating how the use of antibiotics can lead directly to the proliferation of resistance in, and potentially among, pathogens.

Description: Organisms with nonphotosynthetic plastids often retain genomes; their gene contents provide clues as to the functions of these organelles. Yet the functional roles of some retained genes—such as those coding for ATP synthase—remain mysterious. In this study, we report the complete plastid genome and transcriptome data of a nonphotosynthetic diatom and propose that its ATP synthase genes may function in ATP hydrolysis to maintain a proton gradient between thylakoids and stroma, required by the twin arginine translocator (Tat) system for translocation of particular proteins into thylakoids. Given the correlated retention of ATP synthase genes and genes for the Tat system in distantly related nonphotosynthetic plastids, we suggest that this Tat-related role for ATP synthase was a key constraint during parallel loss of photosynthesis in multiple independent lineages of algae/plants.

Description: The brown rat, Rattus norvegicus , is both a notorious pest and a frequently used model in biomedical research. By analyzing genome sequences of 12 wild-caught brown rats from their presumed ancestral range in NE China, along with the sequence of a black rat, Rattus rattus , we investigate the selective and demographic forces shaping variation in the genome. We estimate that the recent effective population size ( N e ) of this species = 1.24 x 105 , based on silent site diversity. We compare patterns of diversity in these genomes with patterns in multiple genome sequences of the house mouse ( Mus musculus castaneus ), which has a much larger N e . This reveals an important role for variation in the strength of genetic drift in mammalian genome evolution. By a Pairwise Sequentially Markovian Coalescent analysis of demographic history, we infer that there has been a recent population size bottleneck in wild rats, which we date to approximately 20,000 years ago. Consistent with this, wild rat populations have experienced an increased flux of mildly deleterious mutations, which segregate at higher frequencies in protein-coding genes and conserved noncoding elements. This leads to negative estimates of the rate of adaptive evolution ( α ) in proteins and conserved noncoding elements, a result which we discuss in relation to the strongly positive estimates observed in wild house mice. As a consequence of the population bottleneck, wild rats also show a markedly slower decay of linkage disequilibrium with physical distance than wild house mice.

Description: Despite much attention, history of sheep ( Ovis aries ) evolution, including its dating, demographic trajectory and geographic spread, remains controversial. To address these questions, we generated 45 complete and 875 partial mitogenomic sequences, and performed a meta-analysis of these and published ovine mitochondrial DNA sequences ( n = 3,229) across Eurasia. We inferred that O. orientalis and O. musimon share the most recent female ancestor with O. aries at approximately 0.790 Ma (95% CI: 0.637–0.934 Ma) during the Middle Pleistocene, substantially predating the domestication event (~8–11 ka). By reconstructing historical variations in effective population size, we found evidence of a rapid population increase approximately 20–60 ka, immediately before the Last Glacial Maximum. Analyses of lineage expansions showed two sheep migratory waves at approximately 4.5–6.8 ka (lineages A and B: ~6.4–6.8 ka; C: ~4.5 ka) across eastern Eurasia, which could have been influenced by prehistoric West–East commercial trade and deliberate mating of domestic and wild sheep, respectively. A continent-scale examination of lineage diversity and approximate Bayesian computation analyses indicated that the Mongolian Plateau region was a secondary center of dispersal, acting as a "transportation hub" in eastern Eurasia: Sheep from the Middle Eastern domestication center were inferred to have migrated through the Caucasus and Central Asia, and arrived in North and Southwest China (lineages A, B, and C) and the Indian subcontinent (lineages B and C) through this region. Our results provide new insights into sheep domestication, particularly with respect to origins and migrations to and from eastern Eurasia.

Description: The frequency of horizontal gene transfer (HGT) in mitochondrial DNA varies substantially. In plants, HGT is relatively common, whereas in animals it appears to be quite rare. It is of considerable importance to understand mitochondrial HGT across the major groups of eukaryotes at a genome-wide level, but so far this has been well studied only in plants. In this study, we generated ten new mitochondrial genome sequences and analyzed 40 mitochondrial genomes from the Saccharomycetaceae to assess the magnitude and nature of mitochondrial HGT in yeasts. We provide evidence for extensive, homologous-recombination-mediated, mitochondrial-to-mitochondrial HGT occurring throughout yeast mitochondrial genomes, leading to genomes that are highly chimeric evolutionarily. This HGT has led to substantial intraspecific polymorphism in both sequence content and sequence divergence, which to our knowledge has not been previously documented in any mitochondrial genome. The unexpectedly high frequency of mitochondrial HGT in yeast may be driven by frequent mitochondrial fusion, relatively low mitochondrial substitution rates and pseudohyphal fusion to produce heterokaryons. These findings suggest that mitochondrial HGT may play an important role in genome evolution of a much broader spectrum of eukaryotes than previously appreciated and that there is a critical need to systematically study the frequency, extent, and importance of mitochondrial HGT across eukaryotes.

Description: Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by X174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between X174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4XG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial XG4G chimera required an exogenous supply of both the X174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the X174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein–protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera’s path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.

Description: Head crests are important display structures in wild bird species and are also common in domesticated lineages. Many breeds of domestic rock pigeon ( Columba livia ) have crests of reversed occipital feathers, and this recessive trait is associated with a nonsynonymous coding mutation in the intracellular kinase domain of EphB2 (Ephrin receptor B2). The domestic ringneck dove ( Streptopelia risoria ) also has a recessive crested morph with reversed occipital feathers, and interspecific crosses between crested doves and pigeons produce crested offspring, suggesting a similar genetic basis for this trait in both species. We therefore investigated EphB2 as a candidate for the head crest phenotype of ringneck doves and identified a nonsynonymous coding mutation in the intracellular kinase domain that is significantly associated with the crested morph. This mutation is over 100 amino acid positions away from the crest mutation found in rock pigeons, yet both mutations are predicted to negatively affect the function of ATP-binding pocket. Furthermore, bacterial toxicity assays suggest that "crest" mutations in both species severely impact kinase activity. We conclude that head crests are associated with different mutations in the same functional domain of the same gene in two different columbid species, thereby representing striking evolutionary convergence in morphology and molecules.

Description: The mitochondrial theory of ageing proposes that the cumulative effect of biochemical damage in mitochondria causes mitochondrial mutations and plays a key role in ageing. Numerous studies have applied comparative approaches to test one of the predictions of the theory: That the rate of mitochondrial mutations is negatively correlated with longevity. Comparative studies face three challenges in detecting correlates of mutation rate: Covariation of mutation rates between species due to ancestry, covariation between life-history traits, and difficulty obtaining accurate estimates of mutation rate. We address these challenges using a novel Poisson regression method to examine the link between mutation rate and lifespan in rockfish ( Sebastes ). This method has better performance than traditional sister-species comparisons when sister species are too recently diverged to give reliable estimates of mutation rate. Rockfish are an ideal model system: They have long life spans with indeterminate growth and little evidence of senescence, which minimizes the confounding tradeoffs between lifespan and fecundity. We show that lifespan in rockfish is negatively correlated to rate of mitochondrial mutation, but not the rate of nuclear mutation. The life history of rockfish allows us to conclude that this relationship is unlikely to be driven by the tradeoffs between longevity and fecundity, or by the frequency of DNA replications in the germline. Instead, the relationship is compatible with the hypothesis that mutation rates are reduced by selection in long-lived taxa to reduce the chance of mitochondrial damage over its lifespan, consistent with the mitochondrial theory of ageing.

Description: Complete genome resequencing of populations holds great promise in deconstructing complex polygenic traits to elucidate molecular and developmental mechanisms of adaptation. Egg size is a classic adaptive trait in insects, birds, and other taxa, but its highly polygenic architecture has prevented high-resolution genetic analysis. We used replicated experimental evolution in Drosophila melanogaster and whole-genome sequencing to identify consistent signatures of polygenic egg-size adaptation. A generalized linear-mixed model revealed reproducible allele frequency differences between replicated experimental populations selected for large and small egg volumes at approximately 4,000 single nucleotide polymorphisms (SNPs). Several hundred distinct genomic regions contain clusters of these SNPs and have lower heterozygosity than the genomic background, consistent with selection acting on polymorphisms in these regions. These SNPs are also enriched among genes expressed in Drosophila ovaries and many of these genes have well-defined functions in Drosophila oogenesis. Additional genes regulating egg development, growth, and cell size show evidence of directional selection as genes regulating these biological processes are enriched for highly differentiated SNPs. Genetic crosses performed with a subset of candidate genes demonstrated that these genes influence egg size, at least in the large genetic background. These findings confirm the highly polygenic architecture of this adaptive trait, and suggest the involvement of many novel candidate genes in regulating egg size.

Description: Although initial studies suggested that Denisovan ancestry was found only in modern human populations from island Southeast Asia and Oceania, more recent studies have suggested that Denisovan ancestry may be more widespread. However, the geographic extent of Denisovan ancestry has not been determined, and moreover the relationship between the Denisovan ancestry in Oceania and that elsewhere has not been studied. Here we analyze genome-wide single nucleotide polymorphism data from 2,493 individuals from 221 worldwide populations, and show that there is a widespread signal of a very low level of Denisovan ancestry across Eastern Eurasian and Native American (EE/NA) populations. We also verify a higher level of Denisovan ancestry in Oceania than that in EE/NA; the Denisovan ancestry in Oceania is correlated with the amount of New Guinea ancestry, but not the amount of Australian ancestry, indicating that recent gene flow from New Guinea likely accounts for signals of Denisovan ancestry across Oceania. However, Denisovan ancestry in EE/NA populations is equally correlated with their New Guinea or their Australian ancestry, suggesting a common source for the Denisovan ancestry in EE/NA and Oceanian populations. Our results suggest that Denisovan ancestry in EE/NA is derived either from common ancestry with, or gene flow from, the common ancestor of New Guineans and Australians, indicating a more complex history involving East Eurasians and Oceanians than previously suspected.

Description: Evidence is mounting that epistasis is widespread among mutations. The cost of carrying two deleterious mutations, or the advantage of acquiring two beneficial alleles, is typically lower that the sum of their individual effects. Much less is known on epistasis between beneficial and deleterious mutations, even though this is key to the amount of genetic hitchhiking that may occur during evolution. This is particularly important in the context of antibiotic resistance: Most resistances are deleterious, but some can be beneficial and remarkably rifampicin resistance can emerge de novo in populations evolving without antibiotics. Here we show pervasive positive pairwise epistasis on Escherichia coli fitness between beneficial mutations, which confer resistance to rifampicin, and deleterious mutations, which confer resistance to streptomycin. We find that 65% of double resistant strains outcompete sensitive bacteria in an environment devoid of antibiotics. Weak beneficial mutations may therefore overcome strong deleterious mutations and can even render double mutants strong competitors.

Description: There is increasing evidence that dosage compensation is not a ubiquitous feature following sex chromosome evolution, especially not in organisms where females are the heterogametic sex, like in birds. Even when it occurs, compensation can be incomplete and limited to dosage-sensitive genes. However, previous work has mainly studied transcriptional regulation of sex-linked genes, which may not reflect expression at the protein level. Here, we used liquid chromatography–tandem mass spectrometry to detect and quantify expressed levels of more than 2,400 proteins in ten different tissues of male and female chicken embryos. For comparison, transcriptome sequencing was performed in the same individuals, five of each sex. The proteomic analysis revealed that dosage compensation was incomplete, with a mean male-to-female (M:F) expression ratio of Z-linked genes of 1.32 across tissues, similar to that at the RNA level (1.29). The mean Z chromosome-to-autosome expression ratio was close to 1 in males and lower than 1 in females, consistent with partly reduced Z chromosome expression in females. Although our results exclude a general mechanism for chromosome-wide dosage compensation at translation, 30% of all proteins encoded from Z-linked genes showed a significant change in the M:F ratio compared with the corresponding ratio at the RNA level. This resulted in a pattern where some genes showed balanced expression between sexes and some close to 2-fold higher expression in males. This suggests that proteomic analyses will be necessary to reveal a more complete picture of gene regulation and sex chromosome evolution.

Description: Flowering time is one of the key determinants of crop adaptation to local environments during domestication. However, the genetic basis underlying flowering time is yet to be elucidated in most cereals. Although staple cereals, such as rice, maize, wheat, barley, and sorghum, have spread and adapted to a wide range of ecological environments during domestication, it is yet to be determined whether they have a common genetic basis for flowering time. In this study, we show, through map-based cloning, that flowering time in sorghum is controlled by a major quantitative trait locus (QTL) Heading Date 1 ( HD1 ), located on chromosome 10. The causal gene encodes the CONSTANS gene family which contains a CCT domain. A 5-bp deletion of a minor allele present in the coding sequence leads to a gene frameshift that delays flowering in sorghum. In contrast, in foxtail millet, association mapping of HD1 showed a common causal site with a splicing variant from "GT" to "AT" that was highly correlated with flowering time. In addition, the rice HD1 gene is known to harbor several causal variants controlling flowering time. These data indicate that the major flowering time QTL HD1 was under parallel domestication in sorghum, foxtail millet, and rice. The pattern of common mixed minor, or even rare, causal alleles in HD1 across different species may be representative of the genetic basis of the domestication syndrome. Furthermore, large DNA sequence analysis of HD1 revealed multiple origins for domesticated sorghum and a single origin for domesticated foxtail millet.

Description: The fundamental asymmetry of female meiosis creates an arena for genetic elements to compete for inclusion in the egg, promoting the selfish evolution of centromere variants that maximize their transmission to the future egg. Such "female meiotic drive" has been hypothesized to explain the paradoxically complex and rapidly evolving nature of centromeric DNA and proteins. Although theoretically widespread, few cases of active drive have been observed, thereby limiting the opportunities to directly assess the impact of centromeric drive on molecular variation at centromeres and binding proteins. Here, we characterize the molecular evolutionary patterns of CENH3 , the centromere-defining histone variant, in Mimulus monkeyflowers, a genus with one of the few known cases of active centromere-associated female meiotic drive. First, we identify a novel duplication of CENH3 in diploid Mimulus , including in lineages with actively driving centromeres. Second, we demonstrate long-term adaptive evolution at several sites in the N-terminus of CENH3 , a region with some meiosis-specific functions that putatively interacts with centromeric DNA. Finally, we infer that the paralogs evolve under different selective regimes; some sites in the N-terminus evolve under positive selection in the pro-orthologs or only one paralog ( CENH3_B ) and the paralogs exhibit significantly different patterns of polymorphism within populations. Our finding of long-term, adaptive evolution at CENH3 in the context of centromere-associated meiotic drive supports an antagonistic, coevolutionary battle for evolutionary dominance between centromeric DNA and binding proteins.

Description: The genomic G+C content of ocean bacteria varies from below 30% to over 60%. This broad range of base composition is likely shaped by distinct mutational processes, recombination, effective population size, and selection driven by environmental factors. A number of studies have hypothesized that depletion of G/C in genomes of marine bacterioplankton cells is an adaptation to the nitrogen-poor pelagic oceans, but they failed to disentangle environmental factors from mutational biases and population history. Here, we reconstructed the evolutionary changes of bases at synonymous sites in genomes of two marine SAR11 populations and a freshwater counterpart with its evolutionary origin rooted in the marine lineage. Although they all have similar genome sizes, DNA repair gene repertoire, and base compositions, there is a stronger bias toward A/T changes, a reduced frequency of nitrogenous amino acids, and an exclusive occurrence of polyamine, opine, and taurine transport systems in the ocean populations, consistent with a greater nitrogen stress in surface oceans compared with freshwater lakes. Furthermore, the ratio of nonsynoymous to synonymous nucleotide diversity is not statistically distinguishable among these populations, suggesting that population history has a limited effect. Taken together, the ecological transition of SAR11 from ocean to freshwater habitats makes nitrogen more available to these organisms, and thus relaxation of purifying selection drove a genome-wide reduction in the frequency of G/C to A/T changes in the freshwater population.

Description: The elevated rate of evolution for genes on sex chromosomes compared with autosomes (Fast-X or Fast-Z evolution) can result either from positive selection in the heterogametic sex or from nonadaptive consequences of reduced relative effective population size. Recent work in birds suggests that Fast-Z of coding sequence is primarily due to relaxed purifying selection resulting from reduced relative effective population size. However, gene sequence and gene expression are often subject to distinct evolutionary pressures; therefore, we tested for Fast-Z in gene expression using next-generation RNA-sequencing data from multiple avian species. Similar to studies of Fast-Z in coding sequence, we recover clear signatures of Fast-Z in gene expression; however, in contrast to coding sequence, our data indicate that Fast-Z in expression is due to positive selection acting primarily in females. In the soma, where gene expression is highly correlated between the sexes, we detected Fast-Z in both sexes, although at a higher rate in females, suggesting that many positively selected expression changes in females are also expressed in males. In the gonad, where intersexual correlations in expression are much lower, we detected Fast-Z for female gene expression, but crucially, not males. This suggests that a large amount of expression variation is sex-specific in its effects within the gonad. Taken together, our results indicate that Fast-Z evolution of gene expression is the product of positive selection acting on recessive beneficial alleles in the heterogametic sex. More broadly, our analysis suggests that the adaptive potential of Z chromosome gene expression may be much greater than that of gene sequence, results which have important implications for the role of sex chromosomes in speciation and sexual selection.

Description: In species with a heterogametic sex, population genetics theory predicts that DNA sequences on the X chromosome can evolve faster than comparable sequences on autosomes. Both neutral and nonneutral evolutionary processes can generate this pattern. Complex traits like gene expression are not predicted to have accelerated evolution by these theories, yet a "faster-X" pattern of gene expression divergence has recently been reported for both Drosophila and mammals. Here, we test the hypothesis that accelerated adaptive evolution of cis -regulatory sequences on the X chromosome is responsible for this pattern by comparing the relative contributions of cis - and trans -regulatory changes to patterns of faster-X expression divergence observed between strains and species of Drosophila with a range of divergence times. We find support for this hypothesis, especially among male-biased genes, when comparing different species. However, we also find evidence that trans -regulatory differences contribute to a faster-X pattern of expression divergence both within and between species. This contribution is surprising because trans -acting regulators of X-linked genes are generally assumed to be randomly distributed throughout the genome. We found, however, that X-linked transcription factors appear to preferentially regulate expression of X-linked genes, providing a potential mechanistic explanation for this result. The contribution of trans -regulatory variation to faster-X expression divergence was larger within than between species, suggesting that it is more likely to result from neutral processes than positive selection. These data show how accelerated evolution of both coding and noncoding sequences on the X chromosome can lead to accelerated expression divergence on the X chromosome relative to autosomes.

Description: Molecular chaperones fold many proteins and their mutated versions in a cell and can sometimes buffer the phenotypic effect of mutations that affect protein folding. Unanswered questions about this buffering include the nature of its mechanism, its influence on the genetic variation of a population, the fitness trade-offs constraining this mechanism, and its role in expediting evolution. Answering these questions is fundamental to understand the contribution of buffering to increase genetic variation and ecological diversification. Here, we performed experimental evolution, genome resequencing, and computational analyses to determine the trade-offs and evolutionary trajectories of Escherichia coli expressing high levels of the essential chaperonin GroEL. GroEL is abundantly present in bacteria, particularly in bacteria with large loads of deleterious mutations, suggesting its role in mutational buffering. We show that groEL overexpression is costly to large populations evolving in the laboratory, leading to groE expression decline within 66 generations. In contrast, populations evolving under the strong genetic drift characteristic of endosymbiotic bacteria avoid extinction or can be rescued in the presence of abundant GroEL. Genomes resequenced from cells evolved under strong genetic drift exhibited significantly higher tolerance to deleterious mutations at high GroEL levels than at native levels, revealing that GroEL is buffering mutations in these cells. GroEL buffered mutations in a highly diverse set of proteins that interact with the environment, including substrate and ion membrane transporters, hinting at its role in ecological diversification. Our results reveal the fitness trade-offs of mutational buffering and how genetic variation is maintained in populations.

Description: Natural selection inference methods often target one mode of selection of a particular age and strength. However, detecting multiple modes simultaneously, or with atypical representations, would be advantageous for understanding a population’s evolutionary history. We have developed an anomaly detection algorithm using distributions of pairwise time to most recent common ancestor (TMRCA) to simultaneously detect multiple modes of natural selection in whole-genome sequences. As natural selection distorts local genealogies in distinct ways, the method uses pairwise TMRCA distributions, which approximate genealogies at a nonrecombining locus, to detect distortions without targeting a specific mode of selection. We evaluate the performance of our method, TSel, for both positive and balancing selection over different time-scales and selection strengths and compare TSel’s performance with that of other methods. We then apply TSel to the Complete Genomics diversity panel, a set of human whole-genome sequences, and recover loci previously inferred to be under positive or balancing selection.

Description: Most studies on the evolution of antibiotic resistance are focused on selection for resistance at lethal antibiotic concentrations, which has allowed the detection of mutant strains that show strong phenotypic traits. However, solely focusing on lethal concentrations of antibiotics narrowly limits our perspective of antibiotic resistance evolution. New high-resolution competition assays have shown that resistant bacteria are selected at relatively low concentrations of antibiotics. This finding is important because sublethal concentrations of antibiotics are found widely in patients undergoing antibiotic therapies, and in nonmedical conditions such as wastewater treatment plants, and food and water used in agriculture and farming. To understand the impacts of sublethal concentrations on selection, we measured 30 adaptive landscapes for a set of TEM β-lactamases containing all combinations of the four amino acid substitutions that exist in TEM-50 for 15 β-lactam antibiotics at multiple concentrations. We found that there are many evolutionary pathways within this collection of landscapes that lead to nearly every TEM-genotype that we studied . While it is known that the pathways change depending on the type of β-lactam, this study demonstrates that the landscapes including fitness optima also change dramatically as the concentrations of antibiotics change. Based on these results we conclude that the presence of multiple concentrations of β-lactams in an environment result in many different adaptive landscapes through which pathways to nearly every genotype are available. Ultimately this may increase the diversity of genotypes in microbial populations.

Description: The availability of extensive databases of crop genome sequences should allow analysis of crop variability at an unprecedented scale, which should have an important impact in plant breeding. However, up to now the analysis of genetic variability at the whole-genome scale has been mainly restricted to single nucleotide polymorphisms (SNPs). This is a strong limitation as structural variation (SV) and transposon insertion polymorphisms are frequent in plant species and have had an important mutational role in crop domestication and breeding. Here, we present the first comprehensive analysis of melon genetic diversity, which includes a detailed analysis of SNPs, SV, and transposon insertion polymorphisms. The variability found among seven melon varieties representing the species diversity and including wild accessions and highly breed lines, is relatively high due in part to the marked divergence of some lineages. The diversity is distributed nonuniformly across the genome, being lower at the extremes of the chromosomes and higher in the pericentromeric regions, which is compatible with the effect of purifying selection and recombination forces over functional regions. Additionally, this variability is greatly reduced among elite varieties, probably due to selection during breeding. We have found some chromosomal regions showing a high differentiation of the elite varieties versus the rest, which could be considered as strongly selected candidate regions. Our data also suggest that transposons and SV may be at the origin of an important fraction of the variability in melon, which highlights the importance of analyzing all types of genetic variability to understand crop genome evolution.

Description: Here, we present a simple, modular and efficient strategy that allows the 3'-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3'-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.

Description: Despite the increasing knowledge about DNA methylation, the understanding of human epigenome evolution is in its infancy. Using whole genome bisulfite sequencing we identified hundreds of differentially methylated regions (DMRs) in humans compared to non-human primates and estimated that ~25% of these regions were detectable throughout several human tissues. Human DMRs were enriched for specific histone modifications and the majority were located distal to transcription start sites, highlighting the importance of regions outside the direct regulatory context. We also found a significant excess of endogenous retrovirus elements in human-specific hypomethylated. We reported for the first time a close interplay between inter-species genetic and epigenetic variation in regions of incomplete lineage sorting, transcription factor binding sites and human differentially hypermethylated regions. Specifically, we observed an excess of human-specific substitutions in transcription factor binding sites located within human DMRs, suggesting that alteration of regulatory motifs underlies some human-specific methylation patterns. We also found that the acquisition of DNA hypermethylation in the human lineage is frequently coupled with a rapid evolution at nucleotide level in the neighborhood of these CpG sites. Taken together, our results reveal new insights into the mechanistic basis of human-specific DNA methylation patterns and the interpretation of inter-species non-coding variation.

Description: The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum . We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.

Description: MicroRNAs are a class of small regulatory RNAs that are generated from primary miRNA (pri-miRNA) transcripts with a stem-loop structure. Accuracy of the processing of pri-miRNA into mature miRNA in plants can be enhanced by SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1). HYL1 activity is regulated by the FIERY2 (FRY2)/RNA polymerase II C-terminal domain phosphatase-like 1 (CPL1). Here, we discover that HIGH OSMOTIC STRESS GENE EXPRESSION 5 (HOS5) and two serine/arginine-rich splicing factors RS40 and RS41, previously shown to be involved in pre-mRNA splicing, affect the biogenesis of a subset of miRNA. These proteins are required for correct miRNA strand selection and the maintenance of miRNA levels. FRY2 dephosphorylates HOS5 whose phosphorylation status affects its subnuclear localization. HOS5 and the RS proteins bind both intronless and intron-containing pri-miRNAs. Importantly, all of these splicing-related factors directly interact with both HYL1 and SE in nuclear splicing speckles. Our results indicate that these splicing factors are directly involved in the biogenesis of a group of miRNA.