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The authors propose that short repeat sequences may play an important role in causing the pervasive clustering of mutations across diverse genomes from prokaryotes to humans., The genome-sequencing gold rush has facilitated the use of comparative genomics to uncover patterns of genome evolution, although their causal mechanisms remain elusive. One such trend, ubiquitous to prokarya and eukarya, is the association of insertion/deletion mutations (indels) with increases in the nucleotide substitution rate extending over hundreds of base pairs. The prevailing hypothesis is that indels are themselves mutagenic agents. Here, we employ population genomics data from Escherichia coli, Saccharomyces paradoxus, and Drosophila to provide evidence suggesting that it is not the indels per se but the sequence in which indels occur that causes the accumulation of nucleotide substitutions. We found that about two-thirds of indels are closely associated with repeat sequences and that repeat sequence abundance could be used to identify regions of elevated sequence diversity, independently of indels. Moreover, the mutational signature of indel-proximal nucleotide substitutions matches that of error-prone DNA polymerases. We propose that repeat sequences promote an increased probability of replication fork arrest, causing the persistent recruitment of error-prone DNA polymerases to specific sequence regions over evolutionary time scales. Experimental measures of the mutation rates of engineered DNA sequences and analyses of experimentally obtained collections of spontaneous mutations provide molecular evidence supporting our hypothesis. This study uncovers a new role for repeat sequences in genome evolution and provides an explanation of how fine-scale sequence contextual effects influence mutation rates and thereby evolution., Author Summary An intriguing observation made during the comparison of genomes is that insertion and deletion mutations (indels) cluster together with nucleotide substitutions. Two (not mutually exclusive) hypotheses have been proposed to explain this phenomenon. The first postulates that an indel mutation causes an increase in the likelihood of the surrounding sequence incurring nucleotide substitutions, while the second claims that the region of DNA in which such a cluster is located is more likely to sustain both indels and substitutions. Here, we present evidence suggesting that the region of DNA, and not the indel, is associated with the accumulation of clusters of mutations over evolutionary time scales. We find that repeat sequences are closely associated with a large proportion of indels and that the abundance of repeat sequences is linked with regions of increased nucleotide diversity. By analysing molecular data and measuring the mutation rates of genes engineered to contain repeats, we find that the mutation rate can be manipulated by the insertion of long repeat sequences. On the basis of these results, we propose a model in which repeat sequences are prone to cause stalling of the high-fidelity DNA polymerase, leading to the recruitment of error-prone repair polymerases which then replicate the surrounding sequence with a higher-than-average error rate.

In a series of publications, I and my colleagues have developed hypotheses for how the evolution of various aspects of genome architecture is expected to proceed under conditions in which the forces of random genetic drift and mutation predominate (e.g., [1]–[15]). These models, collectively referred to below as the mutational-hazard (hereafter, MH) hypothesis, are sometimes represented as neutral models [16], [17], but this is not correct, as the key component of each model is the deleterious mutational consequence of excess DNA. The MH hypothesis is, however, a nonadaptational model, in that it yields expectations on the structure of genomes without invoking external selective forces. It is likely that some aspects of these models will need to be changed as more is learned about the molecular consequences of various aspects of gene structure and the nature of mutation. Such modifications will not alter the need for baseline null hypotheses in attempts to defend adaptive explanations for variation in genomic architecture [9]. Nevertheless, any theory that strives to provide a unifying explanation for diverse sets of genomic observations must be scrutinized extensively from a variety of angles and interpreted in the context of well-established molecular and population-genetic processes. Although I will argue that a recent challenge to the MH hypothesis by Whitney and Garland ([18]; hereafter, WG) contains numerous problems, this exchange may help clarify more broadly misunderstood issues.

The relationships between the levels of transcripts and the levels of the proteins they encode have not been examined comprehensively in mammals, although previous work in plants and yeast suggest a surprisingly modest correlation. We have examined this issue using a genetic approach in which natural variations were used to perturb both transcript levels and protein levels among inbred strains of mice. We quantified over 5,000 peptides and over 22,000 transcripts in livers of 97 inbred and recombinant inbred strains and focused on the 7,185 most heritable transcripts and 486 most reliable proteins. The transcript levels were quantified by microarray analysis in three replicates and the proteins were quantified by Liquid Chromatography–Mass Spectrometry using O(18)-reference-based isotope labeling approach. We show that the levels of transcripts and proteins correlate significantly for only about half of the genes tested, with an average correlation of 0.27, and the correlations of transcripts and proteins varied depending on the cellular location and biological function of the gene. We examined technical and biological factors that could contribute to the modest correlation. For example, differential splicing clearly affects the analyses for certain genes; but, based on deep sequencing, this does not substantially contribute to the overall estimate of the correlation. We also employed genome-wide association analyses to map loci controlling both transcript and protein levels. Surprisingly, little overlap was observed between the protein- and transcript-mapped loci. We have typed numerous clinically relevant traits among the strains, including adiposity, lipoprotein levels, and tissue parameters. Using correlation analysis, we found that a low number of clinical trait relationships are preserved between the protein and mRNA gene products and that the majority of such relationships are specific to either the protein levels or transcript levels. Surprisingly, transcript levels were more strongly correlated with clinical traits than protein levels. In light of the widespread use of high-throughput technologies in both clinical and basic research, the results presented have practical as well as basic implications., Author Summary An old dogma in biology states that, in every cell, the flow of biological information is from DNA to RNA to proteins and that the latter act as a working force to determine the organism's phenotype. This model predicts that changes in DNA that affect the clinical phenotype should also similarly change the cellular levels of RNA and protein levels. In this report, we test this prediction by looking at the concordance between DNA variation in population of mouse inbred strains, the RNA and protein variation in the liver tissue of these mice, and variation in metabolic phenotypes. We show that the relationship between various biological traits is not simple and that there is relatively little concordance of RNA levels and the corresponding protein levels in response to DNA perturbations. In addition, we also find that, surprisingly, metabolic traits correlate better to RNA levels than to protein levels. In light of current efforts in searching for the molecular bases of disease susceptibility in humans, our findings highlight the complexity of information flow that underlies clinical outcomes.

Coordination of fetal maturation with birth timing is essential for mammalian reproduction. In humans, preterm birth is a disorder of profound global health significance. The signals initiating parturition in humans have remained elusive, due to divergence in physiological mechanisms between humans and model organisms typically studied. Because of relatively large human head size and narrow birth canal cross-sectional area compared to other primates, we hypothesized that genes involved in parturition would display accelerated evolution along the human and/or higher primate phylogenetic lineages to decrease the length of gestation and promote delivery of a smaller fetus that transits the birth canal more readily. Further, we tested whether current variation in such accelerated genes contributes to preterm birth risk. Evidence from allometric scaling of gestational age suggests human gestation has been shortened relative to other primates. Consistent with our hypothesis, many genes involved in reproduction show human acceleration in their coding or adjacent noncoding regions. We screened >8,400 SNPs in 150 human accelerated genes in 165 Finnish preterm and 163 control mothers for association with preterm birth. In this cohort, the most significant association was in FSHR, and 8 of the 10 most significant SNPs were in this gene. Further evidence for association of a linkage disequilibrium block of SNPs in FSHR, rs11686474, rs11680730, rs12473870, and rs1247381 was found in African Americans. By considering human acceleration, we identified a novel gene that may be associated with preterm birth, FSHR. We anticipate other human accelerated genes will similarly be associated with preterm birth risk and elucidate essential pathways for human parturition., Children's Discovery Institute, March of Dimes Birth Defects Foundation, National Institute of General Medical Sciences (U.S.) (T32 GM081739), Washington University (Saint Louis, Mo.), Washington University (Saint Louis, Mo.) (Mr. and Mrs. Spencer T. Olin Fellowship for Women in Graduate Study), Sigrid Jusélius stiftelse, Signe and Anne Gyllenberg Foundation, Academy of Finland

Comparative genomic analyses of primates offer considerable potential to define and understand the processes that mold, shape, and transform the human genome. However, primate taxonomy is both complex and controversial, with marginal unifying consensus of the evolutionary hierarchy of extant primate species. Here we provide new genomic sequence (∼8 Mb) from 186 primates representing 61 (∼90%) of the described genera, and we include outgroup species from Dermoptera, Scandentia, and Lagomorpha. The resultant phylogeny is exceptionally robust and illuminates events in primate evolution from ancient to recent, clarifying numerous taxonomic controversies and providing new data on human evolution. Ongoing speciation, reticulate evolution, ancient relic lineages, unequal rates of evolution, and disparate distributions of insertions/deletions among the reconstructed primate lineages are uncovered. Our resolution of the primate phylogeny provides an essential evolutionary framework with far-reaching applications including: human selection and adaptation, global emergence of zoonotic diseases, mammalian comparative genomics, primate taxonomy, and conservation of endangered species., Author Summary Advances in human biomedicine, including those focused on changes in genes triggered or disrupted in development, resistance/susceptibility to infectious disease, cancers, mechanisms of recombination, and genome plasticity, cannot be adequately interpreted in the absence of a precise evolutionary context or hierarchy. However, little is known about the genomes of other primate species, a situation exacerbated by a paucity of nuclear molecular sequence data necessary to resolve the complexities of primate divergence over time. We overcome this deficiency by sequencing 54 nuclear gene regions from DNA samples representing ∼90% of the diversity present in living primates. We conduct a phylogenetic analysis to determine the origin, evolution, patterns of speciation, and unique features in genome divergence among primate lineages. The resultant phylogenetic tree is remarkably robust and unambiguously resolves many long-standing issues in primate taxonomy. Our data provide a strong foundation for illuminating those genomic differences that are uniquely human and provide new insights on the breadth and richness of gene evolution across all primate lineages.

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species., Author Summary The yeast S. cerevisiae has been associated with human activity for thousands of years in industries such as baking, brewing, and winemaking. During this time, humans have effectively domesticated this microorganism, with different industries selecting for specific desirable phenotypic traits. This has resulted in the species S. cerevisiae comprising a genetically diverse collection of individual strains that are often suited to very specific roles (e.g. wine strains produce wine but not beer and vice versa). In order to understand the genetic differences that underpin these diverse industrial characteristics, we have sequenced the genomes of six industrial strains of S. cerevisiae that comprise four strains used in commercial wine production and two strains used in beer brewing. By comparing these genome sequences to existing S. cerevisiae genome sequences from laboratory, pathogenic, bioethanol, and “natural” isolates, we were able to identify numerous genetic differences among these strains including the presence of novel open reading frames and genomic rearrangements, which may provide the basis for the phenotypic differences observed among these strains.

BACKGROUND: The recent completion of the swine genome sequencing project and development of a high density porcine SNP array has made genome-wide association (GWA) studies feasible in pigs. METHODOLOGY/PRINCIPAL FINDINGS: Using Illumina's PorcineSNP60 BeadChip, we performed a pilot GWA study in 820 commercial female pigs phenotyped for backfat, loin muscle area, body conformation in addition to feet and leg (FL) structural soundness traits. A total of 51,385 SNPs were jointly fitted using Bayesian techniques as random effects in a mixture model that assumed a known large proportion (99.5%) of SNPs had zero effect. SNP annotations were implemented through the Sus scrofa Build 9 available from pig Ensembl. We discovered a number of candidate chromosomal regions, and some of them corresponded to QTL regions previously reported. We not only have identified some well-known candidate genes for the traits of interest, such as MC4R (for backfat) and IGF2 (for loin muscle area), but also obtained novel promising genes, including CHCHD3 (for backfat), BMP2 (for loin muscle area, body size and several FL structure traits), and some HOXA family genes (for overall leg action). The candidate regions responsible for body conformation and FL structure soundness did not overlap greatly which implied that these traits were controlled by different genes. Functional clustering analyses classified the genes into categories related to bone and cartilage development, muscle growth and development or the insulin pathway suggesting the traits are regulated by common pathways or gene networks that exert roles at different spatial and temporal stages. CONCLUSIONS/SIGNIFICANCE: This study is one of the earliest GWA reports on important quantitative traits in pigs, and the findings will contribute to the further biological function analysis of the identified candidate genes and potential utilization of them in marker assisted selection.

Recent research has provided mechanistic insight into the important contributions of the gut microbiota to vertebrate biology, but questions remain about the evolutionary processes that have shaped this symbiosis. In the present study, we showed in experiments with gnotobiotic mice that the evolution of Lactobacillus reuteri with rodents resulted in the emergence of host specialization. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and we identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and it identified genomic events that have shaped this process., Author Summary The gastrointestinal microbiota of vertebrates is important for nutrient utilization, resistance against pathogens, and immune maturation of its host, but little is known about the evolutionary relationships between vertebrates and individual bacterial members of these communities. Here we provide robust evidence that the evolution of the gut symbiont Lactobacillus reuteri with vertebrates resulted in the emergence of host specialization. Genomic approaches using a combination of genome sequence comparisons and microarray analysis were used to identify the host-specific genome content in rodent and human strains and the evolutionary events that resulted in host adaptation. The study revealed divergent patterns of genome evolution in rodent and human lineages and a distinct genome inventory in host-restricted sub-populations of L. reuteri that reflected the niche characteristics in the gut of their particular vertebrate hosts. The ecological significance of representative rodent-specific genes was demonstrated in gnotobiotic mice. In conclusion, this work provided evidence that the vertebrate gut symbiont Lactobacillus reuteri, despite the likelihood of horizontal transmission, has remained stably associated with related groups of vertebrate hosts over evolutionary time and has evolved a lifestyle specialized to these host animals.

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment., Author Summary Adaptive radiation is the rapid origination of an array of species by the divergent colonization of disparate ecological niches. In the case of pathogenic bacteria, radiations can lead to the emergence of novel human pathogens. Being divergently adapted to a range of different mammalian hosts, including humans as reservoir or incidental hosts, the genus Bartonella represents a suitable model to study genomic mechanisms underpinning divergent adaptation of pathogens. Here we show that two distinct lineages of Bartonella have radiated in parallel, resulting in two arrays of evolutionary distinct species adapted to overlapping sets of mammalian hosts. Such parallelisms display excellent models to reveal insights into the genetic mechanisms underlying these independent evolutionary processes. Our genome-wide analysis identifies a striking evolutionary parallelism in a horizontally-acquired protein secretion system in the two lineages. The parallel evolutionary trajectory of this system in the two lineages is characterized by the convergent origination of a wide array of adaptive functions dedicated to the cellular interaction within the mammalian hosts. The parallel evolution of the two radiating lineages on the ecological as well as on the molecular level suggests that the horizontal acquisition and the functional diversification of the secretion system display an evolutionary key innovation underlying adaptive evolution.

Genetic predisposition controlled by susceptibility quantitative trait loci (QTLs) contributes to a large proportion of common cancers. Studies of genetics of cancer susceptibility, however, did not address systematically the relationship between susceptibility to cancers in different organs. We present five sets of data on genetic architecture of colon and lung cancer susceptibility in mice, humans and rats. They collectively show that the majority of genes for colon and lung cancer susceptibility are linked pair-wise and are likely identical or related. Four CcS/Dem recombinant congenic strains, each differing from strain BALB/cHeA by a different small random subset of ±12.5% of genes received from strain STS/A, suggestively show either extreme susceptibility or extreme resistance for both colon and lung tumors, which is unlikely if the two tumors were controlled by independent susceptibility genes. Indeed, susceptibility to lung cancer (Sluc) loci underlying the extreme susceptibility or resistance of such CcS/Dem strains, mapped in 226 (CcS-10 x CcS-19)F2 mice, co-localize with susceptibility to colon cancer (Scc) loci. Analysis of additional Sluc loci that were mapped in OcB/Dem strains and Scc loci in CcS/Dem strains, respectively, shows their widespread pair-wise co-localization (P = 0.0036). Finally, the majority of published human and rat colon cancer susceptibility genes map to chromosomal regions homologous to mouse Sluc loci. 12/12 mouse Scc loci, 9/11 human and 5/7 rat colon cancer susceptibility loci are close to a Sluc locus or its homologous site, forming 21 clusters of lung and colon cancer susceptibility genes from one, two or three species. Our data shows that cancer susceptibility QTLs can have much broader biological effects than presently appreciated. It also demonstrates the power of mouse genetics to predict human susceptibility genes. Comparison of molecular mechanisms of susceptibility genes that are organ-specific and those with trans-organ effects can provide a new dimension in understanding individual cancer susceptibility.

In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline., Author Summary In mammals, a subset of genes is expressed from only one chromosomal copy, depending on its parental origin. This process, known as genomic imprinting, results from DNA methylation marks deposited in gametes at regulatory sequences called imprinting control regions (ICRs). Most of the DNA methylation controlling imprinting is established in the oocyte, while very few ICRs are methylated in the sperm. We provided insight into the impact and origins of the parental imbalance in genomic imprinting control. We defined the transcriptome-wide effect of imprinting, during the transition period when the embryo becomes dependent upon maternal resources. We found that maternal ICRs have a vital effect on developmental pathways related to the mother-to-fetus exchanges, while paternal ICRs have a dispersed and non-significant effect at that stage. We evidenced that paternal ICRs are lost at a much faster rate than maternal ICRs during mammalian evolution, probably as a mechanistic consequence of different kinetics of the parental germlines. Our results support the notion that two independent evolutionary forces have led to the numerical and functional dominance of maternal ICRs: a selective advantage of parent-specific regulation of genes important for the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.

Evolution maintains organismal fitness by preserving genomic information. This is widely assumed to involve conservation of specific genomic loci among species. Many genomic encodings are now recognized to integrate small contributions from multiple genomic positions into quantitative dispersed codes, but the evolutionary dynamics of such codes are still poorly understood. Here we show that in yeast, sequences that quantitatively affect nucleosome occupancy evolve under compensatory dynamics that maintain heterogeneous levels of A+T content through spatially coupled A/T-losing and A/T-gaining substitutions. Evolutionary modeling combined with data on yeast polymorphisms supports the idea that these substitution dynamics are a consequence of weak selection. This shows that compensatory evolution, so far believed to affect specific groups of epistatically linked loci like paired RNA bases, is a widespread phenomenon in the yeast genome, affecting the majority of intergenic sequences in it. The model thus derived suggests that compensation is inevitable when evolution conserves quantitative and dispersed genomic functions., Author Summary Purifying selection is a major force in conserving genomic features. It pushes deleterious mutations to extinction while conserving the specific DNA sequence. Here we show that a large proportion of the yeast genome evolves under compensatory dynamics that conserve genomic properties while modifying the genomic sequence. Such compensatory evolution conserves the local G+C content of the genome, which influences nucleosome organization. Since purifying selection is too weak to eliminate every weakly deleterious mutation in nucleosome bound or unbound sequences, the local G+C content is frequently stabilized by compensatory G+C gaining and G+C losing mutations in proximal loci. Theoretical analysis shows that compensatory evolution is inevitable when natural selection is weak and the genomic feature is distributed over many loci. These results imply that sequence conservation may not always be equated with overall selection. They demonstrate that cycles of weakly deleterious substitutions followed by positive selection for corrective mutations, which were so far studied mostly in RNA coding genes, are observed broadly and profoundly affect genome evolution.

Background: Anopheles funestus is one of the primary vectors of human malaria, which causes a million deaths each year in sub-Saharan Africa. Few scientific resources are available to facilitate studies of this mosquito species and relatively little is known about its basic biology and evolution, making development and implementation of novel disease control efforts more difficult. The An. funestus genome has not been sequenced, so in order to facilitate genome-scale experimental biology, we have sequenced the adult female transcriptome of An. funestus from a newly founded colony in Burkina Faso, West Africa, using the Illumina GAIIx next generation sequencing platform. Methodology/Principal Findings: We assembled short Illumina reads de novo using a novel approach involving iterative de novo assemblies and ‘‘target-based’’ contig clustering. We then selected a conservative set of 15,527 contigs through comparisons to four Dipteran transcriptomes as well as multiple functional and conserved protein domain databases. Comparison to the Anopheles gambiae immune system identified 339 contigs as putative immune genes, thus identifying a large portion of the immune system that can form the basis for subsequent studies of this important malaria vector. We identified 5,434 1:1 orthologues between An. funestus and An. gambiae and found that among these 1:1 orthologues, the protein sequence of those with putative immune function were significantly more diverged than the transcriptome as a whole. Short read alignments to the contig set revealed almost 367,000 genetic polymorphisms segregating in the An. funestus colony and demonstrated the utility of the assembled transcriptome for use in RNA-seq based measurements of gene expression. Conclusions/Significance: We developed a pipeline that makes de novo transcriptome sequencing possible in virtually any organism at a very reasonable cost ($6,300 in sequencing costs in our case). We anticipate that our approach could be used to develop genomic resources in a diversity of systems for which full genome sequence is currently unavailable. Our An. funestus contig set and analytical results provide a valuable resource for future studies in this non-model, but epidemiologically critical, vector insect.

A specific and sensitive serodiagnostic test for Mycobacterium ulcerans infection would greatly assist the diagnosis of Buruli ulcer and would also facilitate seroepidemiological surveys. By comparative genomics, we identified 45 potential M. ulcerans specific proteins, of which we were able to express and purify 33 in E. coli. Sera from 30 confirmed Buruli ulcer patients, 24 healthy controls from the same endemic region and 30 healthy controls from a non-endemic region in Benin were screened for antibody responses to these specific proteins by ELISA. Serum IgG responses of Buruli ulcer patients were highly variable, however, seven proteins (MUP045, MUP057, MUL_0513, Hsp65, and the polyketide synthase domains ER, AT propionate, and KR A) showed a significant difference between patient and non-endemic control antibody responses. However, when sera from the healthy control subjects living in the same Buruli ulcer endemic area as the patients were examined, none of the proteins were able to discriminate between these two groups. Nevertheless, six of the seven proteins showed an ability to distinguish people living in an endemic area from those in a non-endemic area with an average sensitivity of 69% and specificity of 88%, suggesting exposure to M. ulcerans. Further validation of these six proteins is now underway to assess their suitability for use in Buruli ulcer seroepidemiological studies. Such studies are urgently needed to assist efforts to uncover environmental reservoirs and understand transmission pathways of the M. ulcerans., Author Summary Buruli ulcer is a slowly progressive but potentially devastating disease of skin and subcutaneous tissue caused by the bacterium Mycobacterium ulcerans. The disease is widespread throughout West and Central Africa, and some countries in the region have established Buruli ulcer control programs. Buruli ulcer is difficult to distinguish from other chronic skin conditions that require different treatments, and there is an urgent need for an accurate point-of-care diagnostic test. In this study, we have used genomic techniques to identify 45 potential M. ulcerans–specific antigens, 33 of which we have been able to produce and purify. We tested these proteins against sera from patients, healthy people living in the same region as the patients and from individuals living in a region with no cases of Buruli ulcer. We found that seven proteins were able to elicit antibody responses that were significantly different between patients and the control subjects from the non-endemic region but not from the healthy individuals in the same Buruli ulcer endemic region. Further analysis showed that six of these M. ulcerans proteins might be useful as markers of exposure to M. ulcerans and could be developed into tools to uncover environmental reservoirs and understand transmission pathways of the bacterium.

The agnostic screening performed by genome-wide association studies (GWAS) has uncovered associations for previously unsuspected genes. Knowledge about the functional role of these genes is crucial and laboratory mouse models can provide such information. Here, we describe a systematic juxtaposition of human GWAS-discovered loci versus mouse models in order to appreciate the availability of mouse models data, to gain biological insights for the role of these genes and to explore the extent of concordance between these two lines of evidence. We perused publicly available data (NHGRI database for human associations and Mouse Genome Informatics database for mouse models) and employed two alternative approaches for cross-species comparisons, phenotype- and gene-centric. A total of 293 single gene-phenotype human associations (262 unique genes and 69 unique phenotypes) were evaluated. In the phenotype-centric approach, we identified all mouse models and related ortholog genes for the 51 human phenotypes with a comparable phenotype in mice. A total of 27 ortholog genes were found to be associated with the same phenotype in humans and mice, a concordance that was significantly larger than expected by chance (p

Natural killer (NK) cells serve essential functions in immunity and reproduction. Diversifying these functions within individuals and populations are rapidly-evolving interactions between highly polymorphic major histocompatibility complex (MHC) class I ligands and variable NK cell receptors. Specific to simian primates is the family of Killer cell Immunoglobulin-like Receptors (KIR), which recognize MHC class I and associate with a range of human diseases. Because KIR have considerable species-specificity and are lacking from common animal models, we performed extensive comparison of the systems of KIR and MHC class I interaction in humans and chimpanzees. Although of similar complexity, they differ in genomic organization, gene content, and diversification mechanisms, mainly because of human-specific specialization in the KIR that recognizes the C1 and C2 epitopes of MHC-B and -C. Humans uniquely focused KIR recognition on MHC-C, while losing C1-bearing MHC-B. Reversing this trend, C1-bearing HLA-B46 was recently driven to unprecedented high frequency in Southeast Asia. Chimpanzees have a variety of ancient, avid, and predominantly inhibitory receptors, whereas human receptors are fewer, recently evolved, and combine avid inhibitory receptors with attenuated activating receptors. These differences accompany human-specific evolution of the A and B haplotypes that are under balancing selection and differentially function in defense and reproduction. Our study shows how the qualitative differences that distinguish the human and chimpanzee systems of KIR and MHC class I predominantly derive from adaptations on the human line in response to selective pressures placed on human NK cells by the competing needs of defense and reproduction., Author Summary Natural killer (NK) cells are versatile lymphocytes that make essential contributions to immune defense and placental reproduction. Essential to NK cell development, diversification and function are variable families of surface receptors that recognize equally variable determinants of polymorphic major histocompatibility complex (MHC) class I molecules, better known as the tissue types matched in clinical organ transplantation. These ligand-receptor interactions evolve rapidly, exhibiting much species specificity and convergent evolution. Consequently, mice represent a poor model, because their receptors are so disparate from the independently evolved human counterparts that are restricted to simian primates. To identify unique and shared aspects of human NK cell biology, we have defined the genomics, population biology, and immunology of variable chimpanzee NK cell receptors and ligands to a level permitting accurate, informed comparison with the well-characterized human system. In both receptors and ligands there are dramatic, qualitative differences between humans and chimpanzees. We show these differences arose during human evolution from the last common human–chimpanzee ancestor, while the chimpanzee system remained relatively stable. That two so closely related species exhibit major differences in NK cell receptors and ligands testifies to the strong and varying selection imposed by the different demands and competing needs of defense and reproduction.

Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10-4, permutation p = 1.0×10-3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10-7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within diseaseassociated loci, thus providing a novel route to aid unravelling common complex diseases. © 2010 Bell et al.

From the ENCODE project, it is realized that almost every base of the entire human genome is transcribed. One class of transcripts resulting from this arises from the conjoined gene, which is formed by combining the exons of two or more distinct (parent) genes lying on the same strand of a chromosome. Only a very limited number of such genes are known, and the definition and terminologies used for them are highly variable in the public databases. In this work, we have computationally identified and manually curated 751 conjoined genes (CGs) in the human genome that are supported by at least one mRNA or EST sequence available in the NCBI database. 353 representative CGs, of which 291 (82%) could be confirmed, were subjected to experimental validation using RT-PCR and sequencing methods. We speculate that these genes are arising out of novel functional requirements and are not merely artifacts of transcription, since more than 70% of them are conserved in other vertebrate genomes. The unique splicing patterns exhibited by CGs reveal their possible roles in protein evolution or gene regulation. Novel CGs, for which no transcript is available, could be identified in 80% of randomly selected potential CG forming regions, indicating that their formation is a routine process. Formation of CGs is not only limited to human, as we have also identified 270 CGs in mouse and 227 in drosophila using our approach. Additionally, we propose a novel mechanism for the formation of CGs. Finally, we developed a database, ConjoinG, which contains detailed information about all the CGs (800 in total) identified in the human genome. In summary, our findings reveal new insights about the functionality of CGs in terms of another possible mechanism for gene regulation and genomic evolution and the mechanism leading to their formation.

BackgroundThe isochore, a large DNA sequence with relatively small GC variance, is one of the most important structures in eukaryotic genomes. Although the isochore has been widely studied in humans and other species, little is known about its distribution in pigs.Principal findingsIn this paper, we construct a map of long homogeneous genome regions (LHGRs), i.e., isochores and isochore-like regions, in pigs to provide an intuitive version of GC heterogeneity in each chromosome. The LHGR pattern study not only quantifies heterogeneities, but also reveals some primary characteristics of the chromatin organization, including the followings: (1) the majority of LHGRs belong to GC-poor families and are in long length; (2) a high gene density tends to occur with the appearance of GC-rich LHGRs; and (3) the density of LINE repeats decreases with an increase in the GC content of LHGRs. Furthermore, a portion of LHGRs with particular GC ranges (50%-51% and 54%-55%) tend to have abnormally high gene densities, suggesting that biased gene conversion (BGC), as well as time- and energy-saving principles, could be of importance to the formation of genome organization.ConclusionThis study significantly improves our knowledge of chromatin organization in the pig genome. Correlations between the different biological features (e.g., gene density and repeat density) and GC content of LHGRs provide a unique glimpse of in silico gene and repeats prediction.

Background The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms. Methods/Principal Findings To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect. Conclusions/Significance Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain.

BackgroundIn spite of extensive research on the effect of mutation and selection on codon usage, a general model of codon usage bias due to mutational bias has been lacking. Because most amino acids allow synonymous GC content changing substitutions in the third codon position, the overall GC bias of a genome or genomic region is highly correlated with GC3, a measure of third position GC content. For individual amino acids as well, G/C ending codons usage generally increases with increasing GC bias and decreases with increasing AT bias. Arginine and leucine, amino acids that allow GC-changing synonymous substitutions in the first and third codon positions, have codons which may be expected to show different usage patterns.Principal findingsIn analyzing codon usage bias in hundreds of prokaryotic and plant genomes and in human genes, we find that two G-ending codons, AGG (arginine) and TTG (leucine), unlike all other G/C-ending codons, show overall usage that decreases with increasing GC bias, contrary to the usual expectation that G/C-ending codon usage should increase with increasing genomic GC bias. Moreover, the usage of some codons appears nonlinear, even nonmonotone, as a function of GC bias. To explain these observations, we propose a continuous-time Markov chain model of GC-biased synonymous substitution. This model correctly predicts the qualitative usage patterns of all codons, including nonlinear codon usage in isoleucine, arginine and leucine. The model accounts for 72%, 64% and 52% of the observed variability of codon usage in prokaryotes, plants and human respectively. When codons are grouped based on common GC content, 87%, 80% and 68% of the variation in usage is explained for prokaryotes, plants and human respectively.ConclusionsThe model clarifies the sometimes-counterintuitive effects that GC mutational bias can have on codon usage, quantifies the influence of GC mutational bias and provides a natural null model relative to which other influences on codon bias may be measured.

BackgroundThe Nuclear Pore Complex (NPC) facilitates molecular trafficking between nucleus and cytoplasm and is an integral feature of the eukaryote cell. It exhibits eight-fold rotational symmetry and is comprised of approximately 30 nucleoporins (Nups) in different stoichiometries. Nups are broadly conserved between yeast, vertebrates and plants, but few have been identified among other major eukaryotic groups.Methodology/principal findingsWe screened for Nups across 60 eukaryote genomes and report that 19 Nups (spanning all major protein subcomplexes) are found in all eukaryote supergroups represented in our study (Opisthokonts, Amoebozoa, Viridiplantae, Chromalveolates and Excavates). Based on parsimony, between 23 and 26 of 31 Nups can be placed in LECA. Notably, they include central components of the anchoring system (Ndc1 and Gp210) indicating that the anchoring system did not evolve by convergence, as has previously been suggested. These results significantly extend earlier results and, importantly, unambiguously place a fully-fledged NPC in LECA. We also test the proposal that transmembrane Pom proteins in vertebrates and yeasts may account for their variant forms of mitosis (open mitoses in vertebrates, closed among yeasts). The distribution of homologues of vertebrate Pom121 and yeast Pom152 is not consistent with this suggestion, but the distribution of fungal Pom34 fits a scenario wherein it was integral to the evolution of closed mitosis in ascomycetes. We also report an updated screen for vesicle coating complexes, which share a common evolutionary origin with Nups, and can be traced back to LECA. Surprisingly, we find only three supergroup-level differences (one gain and two losses) between the constituents of COPI, COPII and Clathrin complexes.Conclusions/significanceOur results indicate that all major protein subcomplexes in the Nuclear Pore Complex are traceable to the Last Eukaryotic Common Ancestor (LECA). In contrast to previous screens, we demonstrate that our conclusions hold regardless of the position of the root of the eukaryote tree.

Correct classification of genes into gene families is important for understanding gene function and evolution. Although gene families of many species have been resolved both computationally and experimentally with high accuracy, gene family classification in most newly sequenced genomes has not been done with the same high standard. This project has been designed to develop a strategy to effectively and accurately classify gene families across genomes. We first examine and compare the performance of computer programs developed for automated gene family classification. We demonstrate that some programs, including the hierarchical average-linkage clustering algorithm MC-UPGMA and the popular Markov clustering algorithm TRIBE-MCL, can reconstruct manual curation of gene families accurately. However, their performance is highly sensitive to parameter setting, i.e. different gene families require different program parameters for correct resolution. To circumvent the problem of parameterization, we have developed a comparative strategy for gene family classification. This strategy takes advantage of existing curated gene families of reference species to find suitable parameters for classifying genes in related genomes. To demonstrate the effectiveness of this novel strategy, we use TRIBE-MCL to classify chemosensory and ABC transporter gene families in C. elegans and its four sister species. We conclude that fully automated programs can establish biologically accurate gene families if parameterized accordingly. Comparative gene family classification finds optimal parameters automatically, thus allowing rapid insights into gene families of newly sequenced species.

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy., Author Summary Leaf-cutter ants form massive subterranean colonies containing millions of workers that harvest hundreds of kilograms of leaves each year. They use these leaves to grow a mutualistic fungus that serves as the colony's primary food source. By farming fungus in specialized garden chambers, these dominant Neotropical herbivores facilitate rapid large-scale plant biomass conversion. Our understanding of this degradation process, and the responsible microbial community, is limited. In this study, we track the degradation of plant polymers in leaf-cutter ant fungus gardens and characterize the microbial community potentially mediating this process. We show that cellulose and hemicelluloses are degraded in the fungus gardens and that a previously unknown microbial community containing a diversity of bacteria is present. Metagenomic analysis of this community's genetic content revealed many genes predicted to encode enzymes capable of degrading plant cell walls. The ability of leaf-cutter ants to maintain an external microbial community with high plant biomass-degrading capacity likely represents a key step in the establishment of these ants as widespread, dominant insect herbivores in the Neotropics. This system is an important model for understanding how microbial communities degrade plant biomass in natural systems and has direct relevancy for bioenergy, given recent interest in cellulosic biofuels.

The combined application of next-generation sequencing platforms has provided an economical approach to unlocking the potential of the turkey genome., A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest., Author Summary In contrast to the compact sequence of viruses and bacteria, determining the complete genome sequence of complex vertebrate genomes can be a daunting task. With the advent of “next-generation” sequencing platforms, it is now possible to rapidly sequence and assemble a vertebrate genome, especially for species for which genomic resources—genetic maps and markers—are currently available. We used a combination of two next-generation sequencing platforms, Roche 454 and Illumina GAII, and unique assembly tools to sequence the genome of the agriculturally important turkey, Meleagris gallopavo. Our draft assembly comprises approximately 1.1 gigabases of which 917 megabytes are assigned to specific chromosomes. Comparisons of the turkey genome sequence with those of the chicken, Gallus gallus, and the zebra finch, Taeniopygia guttata, provide insights into the evolution of the avian lineage. This genome sequence will facilitate discovery of agriculturally important genetic variants.

Mutation is the engine that drives evolution and adaptation forward in that it generates the variation on which natural selection acts. Mutation is a random process that nevertheless occurs according to certain biases. Elucidating mutational biases and the way they vary across species and within genomes is crucial to understanding evolution and adaptation. Here we demonstrate that clonal pathogens that evolve under severely relaxed selection are uniquely suitable for studying mutational biases in bacteria. We estimate mutational patterns using sequence datasets from five such clonal pathogens belonging to four diverse bacterial clades that span most of the range of genomic nucleotide content. We demonstrate that across different types of sites and in all four clades mutation is consistently biased towards AT. This is true even in clades that have high genomic GC content. In all studied cases the mutational bias towards AT is primarily due to the high rate of C/G to T/A transitions. These results suggest that bacterial mutational biases are far less variable than previously thought. They further demonstrate that variation in nucleotide content cannot stem entirely from variation in mutational biases and that natural selection and/or a natural selection-like process such as biased gene conversion strongly affect nucleotide content., Author Summary Natural selection sorts through the variability generated by mutation and biases evolution toward fitter outcomes. However, because mutation is itself not entirely random it can also bias the direction of evolution independently of selection. For instance, it is often assumed that the extreme variation observed in nucleotide content among bacteria (from ∼20% to ∼80% GC) is predominantly driven by extreme differences in mutational biases towards or away from GC. Here, we show that bacterial lineages that recently developed clonal, pathogenic lifestyles evolve under weak selection and that polymorphisms in these bacteria can be used as a fair proxy for mutational spectra. We analyze large sequence datasets from five clonal pathogens in four diverse bacterial clades spanning most of the range of genomic nucleotide content. We find that, surprisingly, mutation is AT-biased in every case to a very similar degree and in each case it is dominated by transitions from C/G to T/A. This demonstrates that mutational biases are far les variable than previously assumed and that variation in bacterial nucleotide content is not due entirely to mutational biases. Rather natural selection or a selection like process such as biased gene conversion strongly affect nucleotide content in bacteria.

Chromosomal location has a significant effect on the evolutionary dynamics of genes involved in sexual dimorphism, impacting both the pattern of sex-specific gene expression and the rate of duplication and protein evolution for these genes. For nearly all non-model organisms, however, knowledge of chromosomal gene content is minimal and difficult to obtain on a genomic scale. In this study, we utilized Comparative Genomic Hybridization (CGH), using probes designed from EST sequence, to identify genes located on the X chromosome of four species in the stalk-eyed fly genus Teleopsis. Analysis of log2 ratio values of female-to-male hybridization intensities from the CGH microarrays for over 3,400 genes reveals a strongly bimodal distribution that clearly differentiates autosomal from X-linked genes for all four species. Genotyping of 33 and linkage mapping of 28 of these genes in Teleopsis dalmanni indicate the CGH results correctly identified chromosomal location in all cases. Syntenic comparison with Drosophila indicates that 90% of the X-linked genes in Teleopsis are homologous to genes located on chromosome 2L in Drosophila melanogaster, suggesting the formation of a nearly complete neo-X chromosome from Muller element B in the dipteran lineage leading to Teleopsis. Analysis of gene movement both relative to Drosophila and within Teleopsis indicates that gene movement is significantly associated with 1) rates of protein evolution, 2) the pattern of gene duplication, and 3) the evolution of eyespan sexual dimorphism. Overall, this study reveals that diopsids are a critical group for understanding the evolution of sex chromosomes within Diptera. In addition, we demonstrate that CGH is a useful technique for identifying chromosomal sex-linkage and should be applicable to other organisms with EST or partial genomic information., Author Summary The distribution and organization of genes on chromosomes vary widely among animals. Chromosomes can change in number and size, as well as gene composition, over short evolutionary time scales. Furthermore, chromosome location can influence how genes are expressed in various tissues and how they evolve. The sex chromosomes, in particular, have a dynamic impact on gene movement, expression, and evolution. Uncovering the chromosomal location of genes has traditionally been difficult for non-model organism species. In this study, we assess sex chromosome linkage using a new method that hybridizes DNA from males and females to probes representing over 3,400 genes in stalk-eyed flies. This technique identifies 533 genes (15%) that are located on the X chromosome with the remaining genes located on two autosomes. Comparison of these genes with their location in Drosophila indicates that the X chromosome in stalk-eyed flies is nearly completely homologous to the autosome 2L in D. melanogaster. This result reveals the formation of a neo-X chromosome in the lineage leading to stalk-eyed flies and indicates that stalk-eyed flies provide a valuable new system to study the origin and evolution of sex chromosomes.

The genomic GC-content of bacteria varies dramatically, from less than 20% to more than 70%. This variation is generally ascribed to differences in the pattern of mutation between bacteria. Here we test this hypothesis by examining patterns of synonymous polymorphism using datasets from 149 bacterial species. We find a large excess of synonymous GC→AT mutations over AT→GC mutations segregating in all but the most AT-rich bacteria, across a broad range of phylogenetically diverse species. We show that the excess of GC→AT mutations is inconsistent with mutation bias, since it would imply that most GC-rich bacteria are declining in GC-content; such a pattern would be unsustainable. We also show that the patterns are probably not due to translational selection or biased gene conversion, because optimal codons tend to be AT-rich, and the excess of GC→AT SNPs is observed in datasets with no evidence of recombination. We therefore conclude that there is selection to increase synonymous GC-content in many species. Since synonymous GC-content is highly correlated to genomic GC-content, we further conclude that there is selection on genomic base composition in many bacteria., Author Summary Shortly after it was proved that DNA was the genetic material it became apparent that organisms, and in particular bacteria, use the four letters of the genetic code to very different extents; the use of G and C varies from less than 20% in some species, to more than 70% in others. This variation in the use of G and C is usually attributed to differences in the pattern of mutation between species. Here we test this hypothesis, and show that, on the contrary, there seems to be pervasive selection on the base composition of the bacterial genome, particularly in GC-rich species. This suggests that many, if not all, sites may be subject to natural selection in many bacteria. Unfortunately, the reason why some bacteria should be selected for high GC-content remains unclear.

Community Page Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum Jayna L. Ditty 1 , Christopher A. Kvaal 2 , Brad Goodner 3 , Sharyn K. Freyermuth 4 , Cheryl Bailey 5 , Robert A. Britton 6 , Stuart G. Gordon 7 , Sabine Heinhorst 8 , Kelynne Reed 9 , Zhaohui Xu 10 , Erin R. Sanders-Lorenz 11 , Seth Axen 12 , Edwin Kim 12 , Mitrick Johns 13 , Kathleen Scott 14 , Cheryl A. Kerfeld 12,15 * 1 Department of Biology, University of St. Thomas, St. Paul, Minnesota, United States of America, 2 Department of Biological Sciences, St. Cloud State University, St. Cloud, Minnesota, United States of America, 3 Department of Biology, Hiram College, Hiram, Ohio, United States of America, 4 Biochemistry Department, University of Missouri- Columbia, Columbia, Missouri, United States of America, 5 Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America, 6 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America, 7 Department of Biology, Presbyterian College, Clinton, South Carolina, United States of America, 8 Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi, United States of America, 9 Biology Department, Austin College, Sherman, Texas, United States of America, 10 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio, United States of America, 11 Department of Microbiology, Immunology and Molecular Genetics, University of California – Los Angeles, Los Angeles, California, United States of America, 12 Department of Energy-Joint Genome Institute, Walnut Creek, California, United States of America, 13 Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America, 14 Department of Integrative Biology, University of South Florida, Tampa, Florida, United States of America, 15 Department of Plant and Microbial Biology, University of California Berkley, Berkeley, California, United States of America Introduction Undergraduate life sciences education needs an overhaul, as clearly described in the National Research Council of the National Academies’ publication BIO 2010: Transforming Undergraduate Education for Future Research Biologists. Among BIO 2010’s top recommendations is the need to involve students in working with real data and tools that reflect the nature of life sciences research in the 21st century [1]. Education research studies support the importance of utilizing primary literature, designing and implementing experiments, and analyzing results in the context of a bona fide scientific question [1–12] in cultivating the analytical skills necessary to become a scientist. Incorporating these basic scientific methodologies in under- graduate education leads to increased undergraduate and post-graduate reten- tion in the sciences [13–16]. Toward this end, many undergraduate teaching orga- nizations offer training and suggestions for faculty to update and improve their teaching approaches to help students learn as scientists, through design and discovery (e.g., Council of Undergraduate Research [www.cur.org] and Project Kaleidoscope [ www.pkal.org]). With the advent of genome sequencing and bioinformatics, many scientists now formulate biological questions and inter- pret research results in the context of genomic information. Just as the use of bioinformatic tools and databases changed the way scientists investigate problems, it must change how scientists teach to create new opportunities for students to gain experiences reflecting the influence of genomics, proteomics, and bioinformatics on modern life sciences research [17–41]. Educators have responded by incorpo- rating bioinformatics into diverse life science curricula [42–44]. While these published exercises in, and guidelines for, bioinformatics curricula are helpful and inspirational, faculty new to the area of bioinformatics inevitably need training in the theoretical underpinnings of the algo- rithms [45]. Moreover, effectively inte- grating bioinformatics into courses or independent research projects requires infrastructure for organizing and assessing student work. Here, we present a new platform for faculty to keep current with the rapidly changing field of bioinfor- matics, the Integrated Microbial Genomes Annotation Collaboration Toolkit (IMG- ACT) (Figure 1). It was developed by instructors from both research-intensive and predominately undergraduate institu- tions in collaboration with the Department of Energy-Joint Genome Institute (DOE- JGI) as a means to innovate and update undergraduate education and faculty de- velopment. The IMG-ACT program pro- vides a cadre of tools, including access to a clearinghouse of genome sequences, bioin- formatics databases, data storage, instruc- tor course management, and student notebooks for organizing the results of their bioinformatic investigations. In the process, IMG-ACT makes it feasible to provide undergraduate research opportu- nities to a greater number and diversity of students, in contrast to the traditional mentor-to-student apprenticeship model for undergraduate research, which can be too expensive and time-consuming to provide for every undergraduate. The IMG-ACT serves as the hub for the network of faculty and students that use the system for microbial genome analysis. Open access of the IMG-ACT infrastructure to participating schools en- sures that all types of higher education institutions can utilize it. With the infra- structure in place, faculty can focus their efforts on the pedagogy of bioinformatics, involvement of students in research, and use of this tool for their own research agenda. What the original faculty mem- bers of the IMG-ACT development team present here is an overview of how the IMG-ACT program has affected our Citation: Ditty JL, Kvaal CA, Goodner B, Freyermuth SK, Bailey C, et al. (2010) Incorporating Genomics and Bioinformatics across the Life Sciences Curriculum. PLoS Biol 8(8): e1000448. doi:10.1371/journal.pbio.1000448 Published August 10, 2010 Copyright: s 2010 Ditty et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: No specific funding was received for this work. The Community Page is a forum for organizations and societies to highlight their efforts to enhance the dissemination and value of scientific knowledge. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: IMG-ACT; Integrated Microbial Genomes Annotation Collaboration Toolkit * E-mail: ckerfeld@lbl.gov PLoS Biology | www.plosbiology.org August 2010 | Volume 8 | Issue 8 | e1000448

The domestic pig is of enormous agricultural significance and valuable models for many human diseases. Information concerning the pig microRNAome (miRNAome) has been long overdue and elucidation of this information will permit an atlas of microRNA (miRNA) regulation functions and networks to be constructed. Here we performed a comprehensive search for porcine miRNAs on ten small RNA sequencing libraries prepared from a mixture of tissues obtained during the entire pig lifetime, from the fetal period through adulthood. The sequencing results were analyzed using mammalian miRNAs, the precursor hairpins (pre-miRNAs) and the first release of the high-coverage porcine genome assembly (Sscrofa9, April 2009) and the available expressed sequence tag (EST) sequences. Our results extend the repertoire of pig miRNAome to 867 pre-miRNAs (623 with genomic coordinates) encoding for 1,004 miRNAs, of which 777 are unique. We preformed real-time quantitative PCR (q-PCR) experiments for selected 30 miRNAs in 47 tissue-specific samples and found agreement between the sequencing and q-PCR data. This broad survey provides detailed information about multiple variants of mature sequences, precursors, chromosomal organization, development-specific expression, and conservation patterns. Our data mining produced a broad view of the pig miRNAome, consisting of miRNAs and isomiRs and a wealth of information of pig miRNA characteristics. These results are prelude to the advancement in pig biology as well the use of pigs as model organism for human biological and biomedical studies.

Bats are reservoirs for a wide range of zoonotic agents including lyssa-, henipah-, SARS-like corona-, Marburg-, Ebola-, and astroviruses. In an effort to survey for the presence of other infectious agents, known and unknown, we screened sera from 16 Pteropus giganteus bats from Faridpur, Bangladesh, using high-throughput pyrosequencing. Sequence analyses indicated the presence of a previously undescribed virus that has approximately 50% identity at the amino acid level to GB virus A and C (GBV-A and -C). Viral nucleic acid was present in 5 of 98 sera (5%) from a single colony of free-ranging bats. Infection was not associated with evidence of hepatitis or hepatic dysfunction. Phylogenetic analysis indicates that this first GBV-like flavivirus reported in bats constitutes a distinct species within the Flaviviridae family and is ancestral to the GBV-A and -C virus clades., Author Summary Bats are important reservoirs for emerging zoonotic viruses with significant impact on human health including lyssaviruses, filoviruses, henipaviruses and coronaviruses. Opportunities for transmission to humans are particularly prominent in countries like Bangladesh, where people live in close association with bats. Whereas previous studies of bats have employed assays that test for known pathogens, we present the first application of an unbiased molecular approach to pathogen discovery in this reservoir for emerging zoonotic disease. Unbiased pyrosequencing of serum from Pteropus giganteus bats enabled identification of a novel flavivirus related to Hepatitis C and GB viruses. Viral nucleic acid was present in 5 of 98 (5%) sera, and in the saliva of one animal. Sequence identification of two strains of the virus, tentatively named GBV-D, suggests P. giganteus as a natural reservoir. Detection of viral nucleic acid in saliva provides a plausible route for zoonotic transmission. Phylogenetic analysis indicates that GBV-D is ancestral to GBV-A and -C, and separate from the recently classified genus Hepacivirus. Our findings provide new insight into the range of known hosts for GB-like viruses and demonstrate the power of unbiased sequencing to characterize the diversity of potentially zoonotic pathogens carried by bats and other reservoirs.

G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint., Author Summary DNA can form structures other than the traditional double helix. The G-quadruplex, a stable four-stranded structure formed by guanine-rich DNA, is one such alternative structure. Sequence motifs with the potential to form G-quadruplex structures (G4 DNA motifs) are found in the genomes of many species. However, since such motifs can occur by chance, it is not known which sequence regions form G-quadruplexes in vivo, and if formed, how they function. Evolutionary conservation of a sequence across species provides evidence of a biologically important function. We found that G4 DNA motifs in S. cerevisiae are more conserved than expected among related fungi, and the patterns of this conservation suggest that many of the motifs form functional G-quadruplexes. We explored potential functions of the G-quadruplex by identifying significant associations of motifs with annotated genome features. Our analysis corroborated and refined previous hypotheses about the importance of G-quadruplexes in telomeres and gene promoters, and suggested intriguing additional roles in double strand break processing and the mitochondria.

In the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB) can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI). However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79–100%) compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16–43%), suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation.

The F. tularensis type A strain FSC198 from Slovakia and a second strain FSC043, which has attenuated virulence, are both considered to be derivatives of the North American F. tularensis type A strain SCHU S4. These strains have been propagated under different conditions: the FSC198 has undergone natural propagation in the environment, while the strain FSC043 has been cultivated on artificial media in laboratories. Here, we have compared the genome sequences of FSC198, FSC043, and SCHU S4 to explore the possibility that the contrasting propagation conditions may have resulted in different mutational patterns. We found four insertion/deletion events (INDELs) in the strain FSC043, as compared to the SCHU S4, while no single nucleotide polymorphisms (SNPs) or variable number of tandem repeats (VNTRs) were identified. This result contrasts with previously reported findings for the strain FSC198, where eight SNPs and three VNTR differences, but no INDELs exist as compared to the SCHU S4 strain. The mutations detected in the laboratory and naturally propagated type A strains, respectively, demonstrate distinct patterns supporting that analysis of mutational spectra might be a useful tool to reveal differences in past growth conditions. Such information may be useful to identify leads in a microbial forensic investigation.

The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across α-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual α-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 α-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator) in the α-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other regulatory networks., Author Summary An important property of living systems is the use of regulatory networks to appropriately program gene expression. Central to the function of regulatory networks are transcription factors that regulate gene expression by binding to specific DNA sequences. Despite the central role of these regulatory networks, the processes driving their organization and evolution across organisms are poorly understood. This paper describes the use of comparative genomics and high-throughput approaches to predict the organization and evolution of transcriptional regulatory networks across a large group of species. We focused on regulatory networks controlling cellular responses to changes in O2 levels because this signal has major consequences on many biological systems. Our analysis predicts that related regulatory networks share a core set of target genes across diverse species while other target genes vary according to the organism's specific lifestyle. Our approach of defining transcriptional regulatory networks across a wide range of organisms should be of general utility to studying similar questions in other systems.

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci., Author Summary Bacterial infections have long been studied using Koch's postulates wherein the paradigm is that a single clone leads to a given infection. Over the past decade, it has become clear that chronic bacterial infections often do not fit this paradigm. Instead these are associated with the presence of multiple strains or species (polyclonal) of bacteria that are organized into highly structured communities, termed biofilms, which can persist in the body and are recalcitrant to antibiotic treatment. In addition, there is extensive evidence that bacteria can incorporate genes from neighboring bacteria into their own genomes. This process can produce new strains and is known as horizontal gene transfer. In this study, we investigated for the first time, the tempo and relevance of gene transfer among bacterial strains of Streptococcus pneumoniae during a naturally occurring chronic childhood infection. We identified extensive gene transfer among multiple infecting strains, by sequencing of isolates recovered sequentially over a seven-month period. This gene transfer may serve as a counterpoint to the host's adaptive immune response and help explain the phenomenon of bacterial persistence, since, as occurs with some chronic viral and parasitic infections, the immune system may become overwhelmed by a set of related strains.

BackgroundCTX-M-producing Escherichia coli strains are regarded as major global pathogens.Methodology/principal findingsThe nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp) from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp) from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla(TEM-1) and bla(CTX-M-15). It shares more than 90% homology with a previously published bla(CTX-M)-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla(TEM-1) and bla(CTX-M-15), were found. Six resistance genes, bla(TEM-1), bla(CTX-M-15), bla(OXA-1), aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla(CTX-M-15)-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla(TEM)-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla(OXA-1), aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids.ConclusionsCollectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of diverse multiresistant plasmids found in clinical Enterobacteriaceae.

It is essential to understand the network of transcription factors controlling self-renewal of human embryonic stem cells (ESCs) and human embryonal carcinoma cells (ECs) if we are to exploit these cells in regenerative medicine regimes. Correlating gene expression levels after RNAi-based ablation of OCT4 function with its downstream targets enables a better prediction of motif-specific driven expression modules pertinent for self-renewal and differentiation of embryonic stem cells and induced pluripotent stem cells. We initially identified putative direct downstream targets of OCT4 by employing CHIP-on-chip analysis. A comparison of three peak analysis programs revealed a refined list of OCT4 targets in the human EC cell line NCCIT, this list was then compared to previously published OCT4 CHIP-on-chip datasets derived from both ES and EC cells. We have verified an enriched POU-motif, discovered by a de novo approach, thus enabling us to define six distinct modules of OCT4 binding and regulation of its target genes. A selection of these targets has been validated, like NANOG, which harbours the evolutionarily conserved OCT4-SOX2 binding motif within its proximal promoter. Other validated targets, which do not harbour the classical HMG motif are USP44 and GADD45G, a key regulator of the cell cycle. Over-expression of GADD45G in NCCIT cells resulted in an enrichment and up-regulation of genes associated with the cell cycle (CDKN1B, CDKN1C, CDK6 and MAPK4) and developmental processes (BMP4, HAND1, EOMES, ID2, GATA4, GATA5, ISL1 and MSX1). A comparison of positively regulated OCT4 targets common to EC and ES cells identified genes such as NANOG, PHC1, USP44, SOX2, PHF17 and OCT4, thus further confirming their universal role in maintaining self-renewal in both cell types. Finally we have created a user-friendly database (http://biit.cs.ut.ee/escd/), integrating all OCT4 and stem cell related datasets in both human and mouse ES and EC cells. In the current era of systems biology driven research, we envisage that our integrated embryonic stem cell database will prove beneficial to the booming field of ES, iPS and cancer research.

Background: The anthrax letter attacks of 2001 highlighted the need for rapid identification of biothreat agents not only for epidemiological surveillance of the intentional outbreak but also for implementing appropriate countermeasures, such as antibiotic treatment, in a timely manner to prevent further casualties. It is clear from the 2001 cases that survival may be markedly improved by administration of antimicrobial therapy during the early symptomatic phase of the illness; i.e., within 3 days of appearance of symptoms. Microbiological detection methods are feasible only for organisms that can be cultured in vitro and cannot detect all genetic modifications with the exception of antibiotic resistance. Currently available immuno or nucleic acid-based rapid detection assays utilize known, organism-specific proteins or genomic DNA signatures respectively. Hence, these assays lack the ability to detect novel natural variations or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic resistant or virulence enhanced Bacillus anthracis, to advise on therapeutic treatments. Methodology/Principal Findings: We show here that the Roche 454-based pyrosequencing can generate whole genome draft sequences of deep and broad enough coverage of a bacterial genome in less than 24 hours. Furthermore, using the unfinished draft sequences, we demonstrate that unbiased identification of known as well as heretofore-unreported genetic modifications that include indels and single nucleotide polymorphisms conferring antibiotic and phage resistances is feasible within the next 12 hours. Conclusions/Significance: Second generation sequencing technologies have paved the way for sequence-based rapid identification of both known and previously undocumented genetic modifications in cultured, conventional and newly emerging biothreat agents. Our findings have significant implications in the context of whole genome sequencing-based routine clinical diagnostics as well as epidemiological surveillance of natural disease outbreaks caused by bacterial and viral agents.

Background The study of large-scale genome structure has revealed patterns suggesting the influence of evolutionary constraints on genome evolution. However, the results of these studies can be difficult to interpret due to the conceptual complexity of the analyses. This makes it difficult to understand how observed statistical patterns relate to the physical distribution of genomic elements. We use a simpler and more intuitive approach to evaluate patterns of genome structure. Methodology/Principal Findings We used randomization tests based on Morisita's Index of aggregation to examine average differences in the distribution of purines and pyrimidines among coding and noncoding regions of 261 chromosomes from 223 microbial genomes representing 21 phylum level groups. Purines and pyrimidines were aggregated in the noncoding DNA of 86% of genomes, but were only aggregated in the coding regions of 52% of genomes. Coding and noncoding DNA differed in aggregation in 94% of genomes. Noncoding regions were more aggregated than coding regions in 91% of these genomes. Genome length appears to limit aggregation, but chromosome length does not. Chromosomes from the same species are similarly aggregated despite substantial differences in length. Aggregation differed among taxonomic groups, revealing support for a previously reported pattern relating genome structure to environmental conditions. Conclusions/Significance Our approach revealed several patterns of genome structure among different types of DNA, different chromosomes of the same genome, and among different taxonomic groups. Similarity in aggregation among chromosomes of varying length from the same genome suggests that individual chromosome structure has not evolved independently of the general constraints on genome structure as a whole. These patterns were detected using simple and readily interpretable methods commonly used in other areas of biology.

Background: Nonrandom distribution of rearrangements is a common feature of eukaryotic chromosomes that is not well understood in terms of genome organization and evolution. In the major African malaria vector Anopheles gambiae, polymorphic inversions are highly nonuniformly distributed among five chromosomal arms and are associated with epidemiologically important adaptations. However, it is not clear whether the genomic content of the chromosomal arms is associated with inversion polymorphism and fixation rates. Methodology/Principal Findings: To better understand the evolutionary dynamics of chromosomal inversions, we created a physical map for an Asian malaria mosquito, Anopheles stephensi, and compared it with the genome of An. gambiae. We also developed and deployed novel Bayesian statistical models to analyze genome landscapes in individual chromosomal arms An. gambiae. Here, we demonstrate that, despite the paucity of inversion polymorphisms on the X chromosome, this chromosome has the fastest rate of inversion fixation and the highest density of transposable elements, simple DNA repeats, and GC content. The highly polymorphic and rapidly evolving autosomal 2R arm had overrepresentation of genes involved in cellular response to stress supporting the role of natural selection in maintaining adaptive polymorphic inversions. In addition, the 2R arm had the highest density of regions involved in segmental duplications that clustered in the breakpoint-rich zone of the arm. In contrast, the slower evolving 2L, 3R, and 3L, arms were enriched with matrixattachment regions that potentially contribute to chromosome stability in the cell nucleus. Conclusions/Significance: These results highlight fundamental differences in evolutionary dynamics of the sex chromosome and autosomes and revealed the strong association between characteristics of the genome landscape and rates of chromosomal evolution. We conclude that a unique combination of various classes of genes and repetitive DNA in each arm, rather than a single type of repetitive element, is likely responsible for arm-specific rates of rearrangements. Published version

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational prediction and functional validation, we have generated a high-resolution genome-wide map of DNA replication origins in Kluyveromyces lactis. Unlike other yeasts or metazoans, K. lactis autonomously replicating sequences (KlARSs) contain a 50 bp consensus motif suggestive of a dimeric structure. This motif is necessary and largely sufficient for initiation and was used to dependably identify 145 of the up to 156 non-repetitive intergenic ARSs projected for the K. lactis genome. Though similar in genome sizes, K. lactis has half as many ARSs as its distant relative S. cerevisiae. Comparative genomic analysis shows that ARSs in K. lactis and S. cerevisiae preferentially localize to non-syntenic intergenic regions, linking ARSs with loci of accelerated evolutionary change., Author Summary DNA replication is an evolutionarily conserved, cell cycle–regulated, spatially and temporally coordinated mechanism in eukaryotes. It is initiated by the binding of the Origin Recognition Complex (ORC) to multiple replication origins. While the ORC is highly conserved, its DNA binding specificity and the primary sequences of replication origins are not. Comparative functional genomics is an obvious approach to addressing questions about the positional conservation and chromosomal determinants of replication origins. However, to date, Saccharomyces cerevisiae is the only eukaryote with a complete genome-wide replication origin map, one which took three decades to compile. We have devised an iterative approach, combining computational prediction and functional validation by direct cloning of replication origins that efficiently identifies a high resolution, near complete repertoire of replication origins in Kluyveromyces lactis. Comparing these two yeast genome maps provides a wealth of information about the DNA elements and positional conservation of replication origins in these two distantly related yeast species. This approach is generally applicable to the construction of high-resolution genome maps of evolutionarily conserved sequences associated with assayable biological functions. Rapid generation of comprehensive functional maps of uncharacterized genomes is critical to whole genome studies of all biological functions.

The understanding of selective constraints affecting genes is a major issue in biology. It is well established that gene expression level is a major determinant of the rate of protein evolution, but the reasons for this relationship remain highly debated. Here we demonstrate that gene expression is also a major determinant of the evolution of gene dosage: the rate of gene losses after whole genome duplications in the Paramecium lineage is negatively correlated to the level of gene expression, and this relationship is not a byproduct of other factors known to affect the fate of gene duplicates. This indicates that changes in gene dosage are generally more deleterious for highly expressed genes. This rule also holds for other taxa: in yeast, we find a clear relationship between gene expression level and the fitness impact of reduction in gene dosage. To explain these observations, we propose a model based on the fact that the optimal expression level of a gene corresponds to a trade-off between the benefit and cost of its expression. This COSTEX model predicts that selective pressure against mutations changing gene expression level or affecting the encoded protein should on average be stronger in highly expressed genes and hence that both the frequency of gene loss and the rate of protein evolution should correlate negatively with gene expression. Thus, the COSTEX model provides a simple and common explanation for the general relationship observed between the level of gene expression and the different facets of gene evolution., Author Summary The analysis of gene evolution is a powerful approach to recognize the genetic features that contribute to the fitness of organisms. It was shown previously that selective constraints on protein sequences increase with expression level. This observation was surprising because there is a priori no reason why lowly expressed genes should be less important than highly expressed genes for the proper function of an organism. Here we show that selective pressure on the evolution of gene dosage, which is another important aspect of gene evolution, is also directly dependent on gene expression level. To explain these observations, we propose a model based on the fact that gene expression is a costly process (notably protein synthesis), so that there is an optimal expression level for each gene corresponding to a trade-off between the benefit and the cost of its expression. This model predicts that selective pressure on gene expression level or on the encoded protein should on average be stronger in highly expressed genes, providing a simple and common explanation for the general relationship observed between gene expression and the different facets of gene evolution.

Background\ud Genome-wide association studies have found type 2 diabetes-associated variants in the HNF1B gene to exhibit reciprocal associations with prostate cancer risk. We aimed to identify whether these variants may have an effect on cancer risk in general versus a specific effect on prostate cancer only.\ud \ud Methodology/Principal Findings\ud In a collaborative analysis, we collected data from GWAS of cancer phenotypes for the frequently reported variants of HNF1B, rs4430796 and rs7501939, which are in linkage disequilibrium (r2 = 0.76, HapMap CEU). Overall, the analysis included 16 datasets on rs4430796 with 19,640 cancer cases and 21,929 controls; and 21 datasets on rs7501939 with 26,923 cases and 49,085 controls. Malignancies other than prostate cancer included colorectal, breast, lung and pancreatic cancers, and melanoma. Meta-analysis showed large between-dataset heterogeneity that was driven by different effects in prostate cancer and other cancers. The per-T2D-risk-allele odds ratios (95% confidence intervals) for rs4430796 were 0.79 (0.76, 0.83)] per G allele for prostate cancer (p

RAD6 is known to suppress duplication-mediated gross chromosomal rearrangements (GCRs) but not single-copy sequence mediated GCRs. Here, we found that the RAD6- and RAD18-dependent post-replication repair (PRR) and the RAD5-, MMS2-, UBC13-dependent error-free PRR branch acted in concert with the replication stress checkpoint to suppress duplication-mediated GCRs formed by homologous recombination (HR). The Rad5 helicase activity, but not its RING finger, was required to prevent duplication-mediated GCRs, although the function of Rad5 remained dependent upon modification of PCNA at Lys164. The SRS2, SGS1, and HCS1 encoded helicases appeared to interact with Rad5, and epistasis analysis suggested that Srs2 and Hcs1 act upstream of Rad5. In contrast, Sgs1 likely functions downstream of Rad5, potentially by resolving DNA structures formed by Rad5. Our analysis is consistent with models in which PRR prevents replication damage from becoming double strand breaks (DSBs) and/or regulates the activity of HR on DSBs., Author Summary Genome instability is a hallmark of many cancers and underlies many inherited disorders that cause a predisposition to cancer. The human genome has many different types of duplicated sequences that can lead to genome instability by recombination-mediated pathways. We previously discovered that duplication-mediated chromosomal rearrangements are suppressed by a number of pathways. Some of these pathways were specific to rearrangements between genomic duplications. Here, we have performed a detailed analysis of pathways dependent upon RAD6, and have discovered that the error-free branch of post-replication repair (PRR) either is as an alternative to homologous recombination or prevents the generation of homologous recombination intermediates. Both of these functions could lead to genomic instability in the context of genomes containing substantial amounts of duplications. The extreme sensitivity of our assay to post-replication repair defects reveals substantial complexity in the interaction of PRR defects, suggesting the presence of many alternative PRR pathways. Together, the results emphasize the importance for appropriately balancing different repair pathways to maintain global genomic stability and highlight a number of defects that could underlie genome instabilities in some cancers.

Much of the eukaryotic genome is known to be mobile, largely due to the movement of transposons and other parasitic elements. Recent work in plants and Drosophila suggests that mobility is also a feature of many nontransposon genes and gene families. Indeed, analysis of the Arabidopsis genome suggested that as many as half of all genes had moved to unlinked positions since Arabidopsis diverged from papaya roughly 72 million years ago, and that these mobile genes tend to fall into distinct gene families. However, the mechanism by which single gene transposition occurred was not deduced. By comparing two closely related species, Arabidopsis thaliana and Arabidopsis lyrata, we sought to determine the nature of gene transposition in Arabidopsis. We found that certain categories of genes are much more likely to have transposed than others, and that many of these transposed genes are flanked by direct repeat sequence that was homologous to sequence within the orthologous target site in A. lyrata and which was predominantly genic in identity. We suggest that intrachromosomal recombination between tandemly duplicated sequences, and subsequent insertion of the circular product, is the predominant mechanism of gene transposition., Author Summary Repetitive DNA, such as satellite repeats and transposons, is ubiquitous throughout the genome. Such repeats have been associated with DNA loss, circle formation, and gene transposition in plants and Drosophila. In this work we suggest that, in plants, one mechanism of gene mobility is intrachromosomal recombination via tandem repeats. In addition, we have demonstrated that the classes of genes that tend to form tandem duplications are more likely to have transposed than other gene classes. We conclude that tandem duplications may particularly facilitate gene excision and may also provide targets for gene insertion.

Previous studies have successfully identified genetic variants in several genes associated with human iris (eye) color; however, they all used simplified categorical trait information. Here, we quantified continuous eye color variation into hue and saturation values using high-resolution digital full-eye photographs and conducted a genome-wide association study on 5,951 Dutch Europeans from the Rotterdam Study. Three new regions, 1q42.3, 17q25.3, and 21q22.13, were highlighted meeting the criterion for genome-wide statistically significant association. The latter two loci were replicated in 2,261 individuals from the UK and in 1,282 from Australia. The LYST gene at 1q42.3 and the DSCR9 gene at 21q22.13 serve as promising functional candidates. A model for predicting quantitative eye colors explained over 50% of trait variance in the Rotterdam Study. Over all our data exemplify that fine phenotyping is a useful strategy for finding genes involved in human complex traits., Author Summary We measured human eye color to hue and saturation values from high-resolution, digital, full-eye photographs of several thousand Dutch Europeans. This quantitative approach, which is extremely cost-effective, portable, and time efficient, revealed that human eye color varies along more dimensions than the one represented by the blue-green-brown categories studied previously. Our work represents the first genome-wide study of quantitative human eye color. We clearly identified 3 new loci, LYST, 17q25.3, TTC3/DSCR9, in contributing to the natural and subtle eye color variation along multiple dimensions, providing new leads towards a more detailed understanding of the genetic basis of human eye color. Our quantitative prediction model explained over 50% of eye color variance, representing the highest accuracy achieved so far in genomic prediction of human complex and quantitative traits, with relevance for future forensic applications.

Gene duplication is a major mechanism facilitating adaptation to changing environments. From recent genomic analyses, the acquisition of zinc hypertolerance and hyperaccumulation characters discriminating Arabidopsis halleri from its zinc sensitive/non-accumulator closest relatives Arabidopsis lyrata and Arabidopsis thaliana was proposed to rely on duplication of genes controlling zinc transport or zinc tolerance. Metal Tolerance Protein 1 (MTP1) is one of these genes. It encodes a Zn2+/H+ antiporter involved in cytoplasmic zinc detoxification and thus in zinc tolerance. MTP1 was proposed to be triplicated in A. halleri, while it is present in single copy in A. thaliana and A. lyrata. Two of the three AhMTP1 paralogues were shown to co-segregate with zinc tolerance in a BC1 progeny from a cross between A. halleri and A. lyrata. In this work, the MTP1 family was characterized at both the genomic and functional levels in A. halleri. Five MTP1 paralogues were found to be present in A. halleri, AhMTP1-A1, -A2, -B, -C, and -D. Interestingly, one of the two newly identified AhMTP1 paralogues was not fixed at least in one A. halleri population. All MTP1s were expressed, but transcript accumulation of the paralogues co-segregating with zinc tolerance in the A. halleri X A. lyrata BC1 progeny was markedly higher than that of the other paralogues. All MTP1s displayed the ability to functionally complement a Saccharomyces cerevisiæ zinc hypersensitive mutant. However, the paralogue showing the least complementation of the yeast mutant phenotype was one of the paralogues co-segregating with zinc tolerance. From our results, the hypothesis that pentaplication of MTP1 could be a major basis of the zinc tolerance character in A. halleri is strongly counter-balanced by the fact that members of the MTP1 family are likely to experience different evolutionary fates, some of which not concurring to increase zinc tolerance., Author Summary Arabidopsis halleri has developed the characters of zinc hypertolerance and hyperaccumulation as compared to its close relatives Arabidopsis thaliana and Arabidopsis lyrata. Different candidate genes were proposed to account for the appearance of these characters in A. halleri. One of them is MTP1 (Metal Tolerance Protein 1), which is involved in cytoplasmic zinc detoxification. We found that A. halleri harbored five gene copies of MTP1, whereas A. thaliana and A. lyrata possess a single copy. It is thus tempting to associate the zinc hypertolerance character of A. halleri to the pentaplication of MTP1. However, we observed that one of the five MTP1 copies is not fixed in a population growing on metal contaminated soil. Also, the different MTP1 genes are markedly differentially expressed in A. halleri, two of them being poorly expressed and repressed in response to zinc constraint. AhMTP1 copies were also demonstrated to display a differential ability to complement the zinc hypersensitivity of a yeast mutant that is dysfunctional in vacuolar zinc transporters. Our findings suggest that different evolutionary fates, some of them not concurring to increase zinc tolerance, are likely to take place for the members of the MTP1 family in A. halleri.

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of diseases. Since the first isolation of C. pneumoniae TWAR in 1965, all human isolates have been essentially clonal, providing little evolutionary insight. To address this gap, we investigated the genetic diversity of 30 isolates from diverse geographical locations, from both human and animal origin (amphibian, reptilian, equine and marsupial). Based on the level of variation that we observed at 23 discreet gene loci, it was clearly evident that the animal isolates were more diverse than the isolates of human origin. Furthermore, we show that C. pneumoniae isolates could be grouped into five major genotypes, A-E, with A, B, D and E genotypes linked by geographical location, whereas genotype C was found across multiple continents. Our evidence strongly supports two separate animal-to-human cross species transfer events in the evolutionary history of this pathogen. The C. pneumoniae human genotype identified in the USA, Canada, Taiwan, Iran, Japan, Korea and Australia (non-Indigenous) most likely originated from a single amphibian or reptilian lineage, which appears to have been previously geographically widespread. We identified a separate human lineage present in two Australian Indigenous isolates (independent geographical locations). This lineage is distinct and is present in Australian amphibians as well as a range of Australian marsupials., Author Summary Chlamydia pneumoniae is an intracellular bacterial pathogen with an extremely diverse host range (humans, amphibians, reptiles and marsupials). We selected 23 target genes in order to investigate genetic diversity: seven of these had been lost or gained by C. pneumoniae, a further six were conserved, four were polymorphic (defined by greater than 20 SNPs per 1 kbp; in this study), and six were truncated or length polymorphic in one strain or the other. Our research highlights that C. pneumoniae animal isolates are much more genetically diverse than C. pneumoniae human isolates, and have crossed the host barrier to humans on at least two occasions. Our study provides new insights into the evolution of this complex pathogen.