Highly Abundant Proteins Are Highly Thermostable.

Highly abundant proteins tend to evolve slowly (a trend called E-R anticorrelation), and a number of hypotheses have been proposed to explain this phenomenon. The misfolding avoidance hypothesis attributes the E-R anticorrelation to the abundance-dependent toxic effects of protein misfolding. To avoid these toxic effects, protein sequences (particularly those of highly expressed proteins) would be under selection to fold properly. One prediction of the misfolding avoidance hypothesis is that highly abundant proteins should exhibit high thermostability (i.e., a highly negative free energy of folding, ΔG). Thus far, only a handful of analyses have tested for a relationship between protein abundance and thermostability, producing contradictory results. These analyses have been limited by 1) the scarcity of ΔG data, 2) the fact that these data have been obtained by different laboratories and under different experimental conditions, 3) the problems associated with using proteins' melting energy (Tm) as a proxy for ΔG, and 4) the difficulty of controlling for potentially confounding variables. Here, we use computational methods to compare the free energy of folding of pairs of human-mouse orthologous proteins with different expression levels. Even though the effect size is limited, the most highly expressed ortholog is often the one with a more negative ΔG of folding, indicating that highly expressed proteins are often more thermostable.

Assessing spatial distribution, genetic variants, and virulence of pathogen Mycoplasma agassizii in threatened Mojave desert tortoises.

Mojave desert tortoises (Gopherus agassizii), a threatened species under the US Endangered Species Act, are long-lived reptiles that experience a chronic respiratory disease. The virulence of primary etiologic agent, Mycoplasma agassizii, remains poorly understood, but it exhibits temporal and geographic variability in causing disease outbreaks in host tortoises. Multiple attempts to culture and characterize the diversity of M. agassizii have had minimal success, even though this opportunistic pathogen chronically persists in nearly every population of Mojave desert tortoises. The current geographic range and the molecular mechanisms of virulence of the type-strain, PS6T, are unknown, and the bacterium is thought to have low-to-moderate virulence. We designed a quantitative polymerase chain reaction (qPCR) targeting three putative virulence genes annotated on the PS6T genome as exo-α-sialidases, enzymes which facilitate growth in many bacterial pathogens. We tested 140 M. agassizii-positive DNA samples collected from 2010 to 2012 across the range of Mojave desert tortoises. We found evidence of multiple-strain infections within hosts. We also found the prevalence of these sialidase-encoding genes to be highest in tortoise populations surrounding southern Nevada, the area from which PS6T was originally isolated. We found a general pattern of loss or reduced presence of sialidase among strains, even within a single host. However, in samples that were positive for any of the putative sialidase genes, one particular gene (528), was positively associated with bacterial loads of M. agassizii and may act as a growth factor for the bacterium. Our results suggest three evolutionary patterns: (1) high levels of variation, possibly due to neutral changes and chronic persistence, (2) a trade-off between moderate virulence and transmission, and (3) selection against virulence in environmental conditions known to be physiologically stressful to the host. Our approach of quantifying genetic variation via qPCR represents a useful model of studying host-pathogen dynamics.

Functional compensation of mouse duplicates by their paralogs expressed in the same tissues.

Analyses in a number of organisms have shown that duplicated genes are less likely to be essential than singletons. This implies that genes can often compensate for the loss of their paralogs. However, it is unclear why the loss of some duplicates can be compensated by their paralogs, whereas the loss of other duplicates cannot. Surprisingly, initial analyses in mice did not detect differences in the essentiality of duplicates and singletons. Only subsequent analyses, using larger gene knockout datasets and controlling for a number of confounding factors, did detect significant differences. Previous studies have not taken into account the tissues in which duplicates are expressed. We hypothesized that in complex organisms, in order for a gene's loss to be compensated by one or more of its paralogs, such paralogs need to be expressed in at least the same set of tissues as the lost gene. To test our hypothesis, we classified mouse duplicates into two categories based on the expression patterns of their paralogs: "compensable duplicates" (those with paralogs expressed in all the tissues in which the gene is expressed) and "non-compensable duplicates" (those whose paralogs are not expressed in all the tissues where the gene is expressed). In agreement with our hypothesis, the essentiality of non-compensable duplicates is similar to that of singletons, whereas compensable duplicates exhibit a substantially lower essentiality. Our results imply that duplicates can often compensate for the loss of their paralogs, but only if they are expressed in the same tissues. Indeed, the compensation ability is more dependent on expression patterns than on protein sequence similarity. The existence of these two kinds of duplicates with different essentialities, which has been overlooked by prior studies, may have hindered the detection of differences between singletons and duplicates.

The final piece of the Triangle of U: Evolution of the tetraploid Brassica carinata genome.

Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species.

The Codon Statistics Database: A Database of Codon Usage Bias.

We present the Codon Statistics Database, an online database that contains codon usage statistics for all the species with reference or representative genomes in RefSeq (over 15,000). The user can search for any species and access two sets of tables. One set lists, for each codon, the frequency, the Relative Synonymous Codon Usage, and whether the codon is preferred. Another set of tables lists, for each gene, its GC content, Effective Number of Codons, Codon Adaptation Index, and frequency of optimal codons. Equivalent tables can be accessed for (1) all nuclear genes, (2) nuclear genes encoding ribosomal proteins, (3) mitochondrial genes, and (4) chloroplast genes (if available in the relevant assembly). The user can also search for any taxonomic group (e.g., "primates") and obtain a table comparing all the species in the group. The database is free to access without registration at http://codonstatsdb.unr.edu.

Extracellular Domains of Transmembrane Proteins Defy the Expression Level-Evolutionary Rate Anticorrelation.

Highly expressed proteins tend to evolve slowly, a trend known as the expression level-rate of evolution (E-R) anticorrelation. Whereas the reasons for this anticorrelation remain unclear, the most influential hypotheses attribute it to highly expressed proteins being subjected to strong selective pressures to avoid misfolding and/or misinteraction. In accordance with these hypotheses, work in our laboratory has recently shown that extracellular (secreted) proteins lack an E-R anticorrelation (or exhibit a weaker than usual E-R anticorrelation). Extracellular proteins are folded inside the endoplasmic reticulum, where enhanced quality control of folding mechanisms exist, and function in the extracellular space, where misinteraction is unlikely to occur or to produce deleterious effects. Transmembrane proteins contain both intracellular domains (which are folded and function in the cytosol) and extracellular domains (which complete their folding in the endoplasmic reticulum and function in the extracellular space). We thus hypothesized that the extracellular domains of transmembrane proteins should exhibit a weaker E-R anticorrelation than their intracellular domains. Our analyses of human, Saccharomyces and Arabidopsis transmembrane proteins allowed us to confirm our hypothesis. Our results are in agreement with models attributing the E-R anticorrelation to the deleterious effects of misfolding and/or misinteraction.

Rates of Protein Evolution across the Marsupial Phylogeny: Heterogeneity and Link to Life-History Traits.

Despite the importance of effective population size (Ne) in evolutionary and conservation biology, it remains unclear what factors have an impact on this quantity. The Nearly Neutral Theory of Molecular Evolution predicts a faster accumulation of deleterious mutations (and thus a higher dN/dS ratio) in populations with small Ne; thus, measuring dN/dS ratios in different groups/species can provide insight into their Ne. Here, we used an exome data set of 1,550 loci from 45 species of marsupials representing 18 of the 22 extant families, to estimate dN/dS ratios across the different branches and families of the marsupial phylogeny. We found a considerable heterogeneity in dN/dS ratios among families and species, which suggests significant differences in their Ne. Furthermore, our multivariate analyses of several life-history traits showed that dN/dS ratios (and thus Ne) are affected by body weight, body length, and weaning age.

Mammals with Small Populations Do Not Exhibit Larger Genomes.

Genome size in cellular organisms varies by six orders of magnitude, yet the cause of this large variation remains unexplained. The influential Drift-Barrier Hypothesis proposes that large genomes tend to evolve in small populations due to inefficient selection. However, to our knowledge no explicit tests of the Drift-Barrier Hypothesis have been reported. We performed the first explicit test, by comparing estimated census population size and genome size in mammals while incorporating potential covariates and the effect of shared evolutionary history. We found a lack of correlation between census population size and genome size among 199 species of mammals. These results suggest that population size is not the predominant factor influencing genome size and that the Drift-Barrier Hypothesis should be considered provisional.

Koala methylomes reveal divergent and conserved DNA methylation signatures of X chromosome regulation.

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is an iconic example of epigenetic regulation. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here, we examine genome-wide signatures of DNA methylation across fives tissues from a male and female koala (Phascolarctos cinereus), and present the first whole-genome, multi-tissue marsupial 'methylome atlas'. Using these novel data, we elucidate divergent versus common features of representative marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in koalas primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx, the primary lncRNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-specific (and presumably inactive X chromosome-specific) expression. Finally, we use the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5%) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.

Mycoplasma agassizii, an opportunistic pathogen of tortoises, shows very little genetic variation across the Mojave and Sonoran Deserts.

Mycoplasma agassizii is a common cause of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii). So far, only two strains of this bacterium have been sequenced, and very little is known about its patterns of genetic diversity. Understanding genetic variability of this pathogen is essential to implement conservation programs for their threatened, long-lived hosts. We used next generation sequencing to explore the genomic diversity of 86 cultured samples of M. agassizii collected from mostly healthy Mojave and Sonoran desert tortoises in 2011 and 2012. All samples with enough sequencing coverage exhibited a higher similarity to M. agassizii strain PS6T (collected in Las Vegas Valley, Nevada) than to strain 723 (collected in Sanibel Island, Florida). All eight genomes with a sequencing coverage over 2x were subjected to multiple analyses to detect single-nucleotide polymorphisms (SNPs). Strikingly, even though we detected 1373 SNPs between strains PS6T and 723, we did not detect any SNP between PS6T and our eight samples. Our whole genome analyses reveal that M. agassizii strain PS6T may be present across a wide geographic extent in healthy Mojave and Sonoran desert tortoises.

Myxosporea (Myxozoa, Cnidaria) Lack DNA Cytosine Methylation.

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.

Richard Dickerson, Molecular Clocks, and Rates of Protein Evolution.

Proteins approximately behave as molecular clocks, accumulating amino acid replacements at a more or less constant rate. Nonetheless, each protein displays a characteristic rate of evolution: whereas some proteins remain largely unaltered over large periods of time, others can rapidly accumulate amino acid replacements. An article by Richard Dickerson, published in the first issue of the Journal of Molecular Evolution (J Mol Evol 1:26-45, 1971), described the first analysis in which the rates of evolution of many proteins were compared, and the differences were interpreted in the light of their function. When comparing the sequences of fibrinopeptides, hemoglobin, and cytochrome c of different species, he observed a linear relationship between the number of amino acid replacements and divergence time. Remarkably, fibrinopeptides had evolved fast, cytochrome c had evolved slowly, and hemoglobin exhibited an intermediate rate of evolution. As the Journal of Molecular Evolution celebrates its 50th anniversary, I highlight this landmark article and reflect on its impact on the field of Molecular Evolution.

Emerging Priorities for Microbiome Research.

Microbiome research has increased dramatically in recent years, driven by advances in technology and significant reductions in the cost of analysis. Such research has unlocked a wealth of data, which has yielded tremendous insight into the nature of the microbial communities, including their interactions and effects, both within a host and in an external environment as part of an ecological community. Understanding the role of microbiota, including their dynamic interactions with their hosts and other microbes, can enable the engineering of new diagnostic techniques and interventional strategies that can be used in a diverse spectrum of fields, spanning from ecology and agriculture to medicine and from forensics to exobiology. From June 19-23 in 2017, the NIH and NSF jointly held an Innovation Lab on Quantitative Approaches to Biomedical Data Science Challenges in our Understanding of the Microbiome. This review is inspired by some of the topics that arose as priority areas from this unique, interactive workshop. The goal of this review is to summarize the Innovation Lab's findings by introducing the reader to emerging challenges, exciting potential, and current directions in microbiome research. The review is broken into five key topic areas: (1) interactions between microbes and the human body, (2) evolution and ecology of microbes, including the role played by the environment and microbe-microbe interactions, (3) analytical and mathematical methods currently used in microbiome research, (4) leveraging knowledge of microbial composition and interactions to develop engineering solutions, and (5) interventional approaches and engineered microbiota that may be enabled by selectively altering microbial composition. As such, this review seeks to arm the reader with a broad understanding of the priorities and challenges in microbiome research today and provide inspiration for future investigation and multi-disciplinary collaboration.

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.

Molecular Chaperones Accelerate the Evolution of Their Protein Clients in Yeast.

Protein stability is a major constraint on protein evolution. Molecular chaperones, also known as heat-shock proteins, can relax this constraint and promote protein evolution by diminishing the deleterious effect of mutations on protein stability and folding. This effect, however, has only been stablished for a few chaperones. Here, we use a comprehensive chaperone-protein interaction network to study the effect of all yeast chaperones on the evolution of their protein substrates, that is, their clients. In particular, we analyze how yeast chaperones affect the evolutionary rates of their clients at two very different evolutionary time scales. We first study the effect of chaperone-mediated folding on protein evolution over the evolutionary divergence of Saccharomyces cerevisiae and S. paradoxus. We then test whether yeast chaperones have left a similar signature on the patterns of standing genetic variation found in modern wild and domesticated strains of S. cerevisiae. We find that genes encoding chaperone clients have diverged faster than genes encoding non-client proteins when controlling for their number of protein-protein interactions. We also find that genes encoding client proteins have accumulated more intraspecific genetic diversity than those encoding non-client proteins. In a number of multivariate analyses, controlling by other well-known factors that affect protein evolution, we find that chaperone dependence explains the largest fraction of the observed variance in the rate of evolution at both evolutionary time scales. Chaperones affecting rates of protein evolution mostly belong to two major chaperone families: Hsp70s and Hsp90s. Our analyses show that protein chaperones, by virtue of their ability to buffer destabilizing mutations and their role in modulating protein genotype-phenotype maps, have a considerable accelerating effect on protein evolution.

What lies beneath? Molecular evolution during the radiation of caecilian amphibians.

BACKGROUND: Evolution leaves an imprint in species through genetic change. At the molecular level, evolutionary changes can be explored by studying ratios of nucleotide substitutions. The interplay among molecular evolution, derived phenotypes, and ecological ranges can provide insights into adaptive radiations. Caecilians (order Gymnophiona), probably the least known of the major lineages of vertebrates, are limbless tropical amphibians, with adults of most species burrowing in soils (fossoriality). This enigmatic order of amphibians are very distinct phenotypically from other extant amphibians and likely from the ancestor of Lissamphibia, but little to nothing is known about the molecular changes underpinning their radiation. We hypothesised that colonization of various depths of tropical soils and of freshwater habitats presented new ecological opportunities to caecilians. RESULTS: A total of 8540 candidate groups of orthologous genes from transcriptomic data of five species of caecilian amphibians and the genome of the frog Xenopus tropicalis were analysed in order to investigate the genetic machinery behind caecilian diversification. We found a total of 168 protein-coding genes with signatures of positive selection at different evolutionary times during the radiation of caecilians. The majority of these genes were related to functional elements of the cell membrane and extracellular matrix with expression in several different tissues. The first colonization of the tropical soils was connected to the largest number of protein-coding genes under positive selection in our analysis. From the results of our study, we highlighted molecular changes in genes involved in perception, reduction-oxidation processes, and aging that likely were involved in the adaptation to different soil strata. CONCLUSIONS: The genes inferred to have been under positive selection provide valuable insights into caecilian evolution, potentially underpin adaptations of caecilians to their extreme environments, and contribute to a better understanding of fossorial adaptations and molecular evolution in vertebrates.

Codon Usage Differences among Genes Expressed in Different Tissues of Drosophila melanogaster.

Codon usage patterns are affected by both mutational biases and translational selection. The frequency at which each codon is used in the genome is directly linked to the cellular concentrations of their corresponding tRNAs. Transfer RNA abundances-as well as the abundances of other potentially relevant factors, such as RNA-binding proteins-may vary across different tissues, making it possible that genes expressed in different tissues are subject to different translational selection regimes, and thus differ in their patterns of codon usage. These differences, however, are poorly understood, having been studied only in Arabidopsis, rice and human, with controversial results in human. Drosophila melanogaster is a suitable model organism to study tissue-specific codon adaptation given its large effective population size. Here, we compare 2,046 genes, each expressed specifically in one tissue of D. melanogaster. We show that genes expressed in different tissues exhibit significant differences in their patterns of codon usage, and that these differences are only partially due to differences in GC content, expression levels, or protein lengths. Remarkably, these differences are stronger when analyses are restricted to highly expressed genes. Our results strongly suggest that genes expressed in different tissues are subject to different regimes of translational selection.

N-glycoproteins exhibit a positive expression level-evolutionary rate correlation.

The different proteins of any proteome evolve at enormously different rates. One of the primary factors influencing rates of protein evolution is expression level, with highly expressed proteins tending to evolve at slow rates. This phenomenon, known as the expression level-evolutionary rate (E-R) anticorrelation, has been attributed to the abundance-dependent deleterious effects of misfolding or misinteraction. We have recently shown that secreted proteins either lack an E-R anticorrelation or exhibit a significantly reduced E-R anticorrelation. This effect may be due to the strict quality control to which secreted proteins are subject in the endoplasmic reticulum (which is expected to reduce the rate of misfolding and its deleterious effects) or to their extracellular location (expected to reduce the rate of misinteraction and its deleterious effects). Among secreted proteins, N-glycosylated ones are under particularly strong quality control. Here, we investigate how N-linked glycosylation affects the E-R anticorrelation. Strikingly, we observe a positive E-R correlation among N-glycosylated proteins. That is, N-glycoproteins that are highly expressed evolve at faster rates than lowly expressed N-glycoproteins, in contrast to what is observed among intracellular proteins.

Proteins of generalist and specialist pathogens differ in their amino acid composition.

Pathogens differ in their host specificities, with species infecting a unique host (specialist pathogens) and others having a wide host range (generalists). Molecular determinants of pathogen's host range remain poorly understood. Secreted proteins of generalist pathogens are expected to have a broader range of intermolecular interactions (i.e., higher promiscuity) compared with their specialist counterparts. We hypothesize that this increased promiscuity of generalist secretomes may be based on an elevated content of primitive amino acids and intrinsically disordered regions, as these features are known to increase protein flexibility and interactivity. Here, we measure the proportion of primitive amino acids and percentage of intrinsically disordered residues in secreted, membrane, and cytoplasmic proteins from pathogens with different host specificity. Supporting our prediction, there is a significant general enrichment for primitive amino acids and intrinsically disordered regions in proteins from generalists compared to specialists, particularly among secreted proteins in prokaryotes. Our findings support our hypothesis that secreted proteins' amino acid composition and disordered content influence the pathogens' host range.