Comparative sequencing provides insights about the structure and conservation of marsupial and monotreme genomes

Sequencing and comparative analyses of genomes from multiple vertebrates are providing insights about the genetic basis for biological diversity. To date, these efforts largely have focused on eutherian mammals, chicken, and fish. In this article, we describe the generation and study of genomic sequences from noneutherian mammals, a group of species occupying unusual phylogenetic positions. A large sequence data set (totaling >5 Mb) was generated for the same orthologous region in three marsupial (North American opossum, South American opossum, and Australian tammar wallaby) and one monotreme (platypus) genomes. These ancient mammalian genomes are characterized by unusual architectural features with respect to G + C and repeat content, as well as compression relative to human. Approximately 14% and 34% of the human sequence forms alignments with the orthologous sequence from platypus and the marsupials, respectively; these numbers are distinctly lower than that observed with nonprimate eutherian mammals (45-70%). The alignable sequences between human and each marsupial species are not completely overlapping (only 80% common to all three species) nor are the platypus-alignable sequences completely contained within the marsupial-alignable sequences. Phylogenetic analysis of synonymous coding positions reveals that platypus has a notably long branch length, with the human-platypus substitution rate being on average 55% greater than that seen with human-marsupial pairs. Finally, analyses of the major mammalian lineages reveal distinct patterns with respect to the common presence of evolutionarily conserved vertebrate sequences. Our results confirm that genomic sequence from noneutherian mammals can contribute uniquely to unraveling the functional and evolutionary histories of the mammalian genome.     

Dynamics of a bacterial multidrug ABC transporter in the inward- and outward-facing conformations

The study of membrane proteins remains a challenging task, and approaches to unravel their dynamics are scarce. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry to probe the motions of a bacterial multidrug ATP-binding cassette (ABC) transporter, BmrA, in the inward-facing (resting state) and outward-facing (ATP-bound) conformations. Trypsin digestion and global or local HDX support the transition between inward- and outward-facing conformations during the catalytic cycle of BmrA. However, in the resting state, peptides from the two intracellular domains, especially ICD2, show a much faster HDX than in the closed state. This shows that these two subdomains are very flexible in this conformation. Additionally, molecular dynamics simulations suggest a large fluctuation of the Cα positions from ICD2 residues in the inward-facing conformation of a related transporter, MsbA. These results highlight the unexpected flexibility of ABC exporters in the resting state and underline the power of HDX coupled to mass spectrometry to explore conformational changes and dynamics of large membrane proteins.     

Functional characterization of piggyBat from the bat Myotis lucifugus unveils an active mammalian DNA transposon

A revelation of the genomic age has been the contributions of the mobile DNA segments called transposable elements to chromosome structure, function, and evolution in virtually all organisms. Substantial fractions of vertebrate genomes derive from transposable elements, being dominated by retroelements that move via RNA intermediates. Although many of these elements have been inactivated by mutation, several active retroelements remain. Vertebrate genomes also contain substantial quantities and a high diversity of cut-and-paste DNA transposons, but no active representative of this class has been identified in mammals. Here we show that a cut-and-paste element called piggyBat, which has recently invaded the genome of the little brown bat (Myotis lucifugus) and is a member of the piggyBac superfamily, is active in its native form in transposition assays in bat and human cultured cells, as well as in the yeast Saccharomyces cerevisiae. Our study suggests that some DNA transposons are still actively shaping some mammalian genomes and reveals an unprecedented opportunity to study the mechanism, regulation, and genomic impact of cut-and-paste transposition in a natural mammalian host.     

APOBECs and Herpesviruses

The apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded (ss)DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes—APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new pathogen-host conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation.     

Three-dimensional structure of the adenine-specific DNA methyltransferase M.Taq I in complex with the cofactor S-adenosylmethionine.

The Thermus aquaticus DNA methyltransferase M.Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of --CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethionine shows alpha/beta folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide cleft suitable to accommodate the DNA substrate. The N-terminal domain is dominated by a nine-stranded beta-sheet; it contains the two conserved segments typical for N-methyltransferases which form a pocket for cofactor binding. The C-terminal domain is formed by four small beta-sheets and alpha-helices. The three-dimensional folding of M.Taq I is similar to that of the cytosine-specific Hha I methyltransferase, where the large beta-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.     

Diversity and evolution of secondary metabolism in the marine actinomycete genus Salinispora

Access to genome sequence data has challenged traditional natural product discovery paradigms by revealing that the products of most bacterial biosynthetic pathways have yet to be discovered. Despite the insight afforded by this technology, little is known about the diversity and distributions of natural product biosynthetic pathways among bacteria and how they evolve to generate structural diversity. Here we analyze genome sequence data derived from 75 strains of the marine actinomycete genus Salinispora for pathways associated with polyketide and nonribosomal peptide biosynthesis, the products of which account for some of today’s most important medicines. The results reveal high levels of diversity, with a total of 124 pathways identified and 229 predicted with continued sequencing. Recent horizontal gene transfer accounts for the majority of pathways, which occur in only one or two strains. Acquired pathways are incorporated into genomic islands and are commonly exchanged within and between species. Acquisition and transfer events largely involve complete pathways, which subsequently evolve by gene gain, loss, and duplication followed by divergence. The exchange of similar pathway types at the precise chromosomal locations in different strains suggests that the mechanisms of integration include pathway-level homologous recombination. Despite extensive horizontal gene transfer there is clear evidence of species-level vertical inheritance, supporting the concept that secondary metabolites represent functional traits that help define Salinispora species. The plasticity of the Salinispora secondary metabolome provides an effective mechanism to maximize population-level secondary metabolite diversity while limiting the number of pathways maintained within any individual genome.Microbial secondary metabolites have long benefited human health and industry. They include important pharmaceutical agents such as the antibiotic penicillin, the anticancer agent vancomycin, and the immunosuppressant rapamycin among the more than 20 thousand biologically active microbial natural products reported as of 2002 (1). Secondary metabolites also have important ecological roles for the organisms that produce them, particularly in terms of nutrient acquisition, chemical communication, and defense (2). Many of these compounds are the products of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) pathways or hybrids thereof. These pathways are generally organized into gene clusters that can exceed 100 kb and include regulatory, resistance, and transport elements (3), thus making them well-suited for horizontal gene transfer (HGT) (4, 5). The architectures and functional attributes of PKS and NRPS genes have been reviewed in detail (3, 6, 7) and account for much of the structural diversity that is the hallmark of microbial natural products. Remarkably, PKS and NRPS enzymes build these complex secondary metabolites via the controlled assembly of simple biosynthetic building blocks such as acetate, propionate, and amino acids. These building blocks are incorporated in a combinatorial fashion via a series of sequential chemical condensation reactions encoded by ketosynthase (KS) and condensation (C) domains within PKS and NRPS genes, respectively (3).The pathways responsible for secondary metabolite biosynthesis are among the most rapidly evolving genetic elements known (5). It has been shown that gene duplication, loss, and HGT have all played important roles in the distribution of PKSs among microbes (8, 9). Changes within PKS and NRPS genes also include mutation, domain rearrangement, and module duplication (5), all of which can account for the generation of new small-molecule diversity. The evolutionary histories of specific PKS and NRPS domains have proven particularly informative, with KS and C domains providing insight into enzyme architecture and function (10, 11). These studies have helped establish the extensive nature of HGT among biosynthetic genes (4, 12), which is reflected in the incongruence between PKS and NRPS gene phylogenies and those of the organisms in which they reside (13). Although resolving the evolutionary histories of entire pathways remains more challenging than individual genes or domains, comparative analyses of biosynthetic gene clusters have proven useful for the identification of pathway boundaries (14).The exchange of PKS and NRPS pathways by HGT confounds the relationships between taxonomy and secondary metabolite production. This may in part explain the historical reliance on chance for the discovery of natural product drug leads from chemically prolific but taxonomically complex taxa such as the genus Streptomyces. Genome sequencing has changed the playing field by providing bioinformatic opportunities to “mine” the biosynthetic potential of strains before chemical analysis and to target the products of specific pathways that are predicted to yield compounds of interest (15). Sequence-based methodologies not only hold great promise for natural product discovery; they are providing a wealth of information that will ultimately improve our understanding of pathway diversity and distributions and the evolutionary events that generate new chemical diversity.Here we report the analysis of PKS and NRPS biosynthetic gene clusters in 75 Salinispora genome sequences. This obligate marine actinomycete is composed of three closely related species (16, 17) that are clearly delineated using phylogenetic approaches (18). Salinispora spp. share 99% 16S rRNA gene sequence identity (19), thus making them more narrowly defined than many taxa, which can include up to 3% sequence divergence (20). Salinispora spp. are a rich source of secondary metabolites, including salinosporamide A (21), which has undergone a series of phase I clinical trials for the treatment of cancer (22). They devote ca. 10% of their genomic content to secondary metabolism (23, 24) and represent a tractable model with which to address correlations between fine-scale molecular systematics and secondary metabolite production (25). The results presented here describe the diversity and distributions of biosynthetic pathways among a closely related group of bacteria and reveal high levels of pathway acquisition via horizontal gene transfer, with more than half of the pathways occurring in only one or two strains. The data provide evidence of the evolutionary mechanisms that generate new pathway diversity and a striking example of the plasticity of the bacterial secondary metabolome.     

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.     

Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501

The capacity to fix nitrogen is widely distributed in phyla of Bacteria and Archaea but has long been considered to be absent from the Pseudomonas genus. We report here the complete genome sequencing of nitrogen-fixing root-associated Pseudomonas stutzeri A1501. The genome consists of a single circular chromosome with 4,567,418 bp. Comparative genomics revealed that, among 4,146 protein-encoding genes, 1,977 have orthologs in each of the five other Pseudomonas representative species sequenced to date. The genome contains genes involved in broad utilization of carbon sources, nitrogen fixation, denitrification, degradation of aromatic compounds, biosynthesis of polyhydroxybutyrate, multiple pathways of protection against environmental stress, and other functions that presumably give A1501 an advantage in root colonization. Genetic information on synthesis, maturation, and functioning of nitrogenase is clustered in a 49-kb island, suggesting that this property was acquired by lateral gene transfer. New genes required for the nitrogen fixation process have been identified within the nif island. The genome sequence offers the genetic basis for further study of the evolution of the nitrogen fixation property and identification of rhizosphere competence traits required in the interaction with host plants; moreover, it opens up new perspectives for wider application of root-associated diazotrophs in sustainable agriculture.     

Infectious Bronchitis Virus: A Comprehensive Multilocus Genomic Analysis to Compare DMV/1639 and QX Strains

Infectious bronchitis virus (IBV) is a highly variable RNA virus that affects chickens worldwide. Due to its inherited tendency to suffer point mutations and recombination events during viral replication, emergent IBV strains have been linked to nephropathogenic and reproductive disease that are more severe than typical respiratory disease, leading, in some cases, to mortality, severe production losses, and/or unsuccessful vaccination. QX and DMV/1639 strains are examples of the above-mentioned IBV evolutionary pathway and clinical outcome. In this study, our purpose was to systematically compare whole genomes of QX and DMV strains looking at each IBV gene individually. Phylogenetic analyses and amino acid site searches were performed in datasets obtained from GenBank accounting for all IBV genes and using our own relevant sequences as a basis. The QX dataset studied is more genetically diverse than the DMV dataset, partially due to the greater epidemiological diversity within the five QX strains used as a basis compared to the four DMV strains from our study. Historically, QX strains have emerged and spread earlier than DMV strains in Europe and Asia. Consequently, there are more QX sequences deposited in GenBank than DMV strains, assisting in the identification of a larger pool of QX strains. It is likely that a similar evolutionary pattern will be observed among DMV strains as they develop and spread in North America.     

Position-dependent correlations between DNA methylation and the evolutionary rates of mammalian coding exons

DNA cytosine methylation is a central epigenetic marker that is usually mutagenic and may increase the level of sequence divergence. However, methylated genes have been reported to evolve more slowly than unmethylated genes. Hence, there is a controversy on whether DNA methylation is correlated with increased or decreased protein evolutionary rates. We hypothesize that this controversy has resulted from the differential correlations between DNA methylation and the evolutionary rates of coding exons in different genic positions. To test this hypothesis, we compare human–mouse and human–macaque exonic evolutionary rates against experimentally determined single-base resolution DNA methylation data derived from multiple human cell types. We show that DNA methylation is significantly related to within-gene variations in evolutionary rates. First, DNA methylation level is more strongly correlated with C-to-T mutations at CpG dinucleotides in the first coding exons than in the internal and last exons, although it is positively correlated with the synonymous substitution rate in all exon positions. Second, for the first exons, DNA methylation level is negatively correlated with exonic expression level, but positively correlated with both nonsynonymous substitution rate and the sample specificity of DNA methylation level. For the internal and last exons, however, we observe the opposite correlations. Our results imply that DNA methylation level is differentially correlated with the biological (and evolutionary) features of coding exons in different genic positions. The first exons appear more prone to the mutagenic effects, whereas the other exons are more influenced by the regulatory effects of DNA methylation.     

Leafy and weedy seadragon genomes connect genic and repetitive DNA features to the extravagant biology of syngnathid fishes

Seadragons are a remarkable lineage of teleost fishes in the family Syngnathidae, renowned for having evolved male pregnancy. Comprising three known species, seadragons are widely recognized and admired for their fantastical body forms and coloration, and their specific habitat requirements have made them flagship representatives for marine conservation and natural history interests. Until recently, a gap has been the lack of significant genomic resources for seadragons. We have produced gene-annotated, chromosome-scale genome models for the leafy and weedy seadragon to advance investigations of evolutionary innovation and elaboration of morphological traits in seadragons as well as their pipefish and seahorse relatives. We identified several interesting features specific to seadragon genomes, including divergent noncoding regions near a developmental gene important for integumentary outgrowth, a high genome-wide density of repetitive DNA, and recent expansions of transposable elements and a vesicular trafficking gene family. Surprisingly, comparative analyses leveraging the seadragon genomes and additional syngnathid and outgroup genomes revealed striking, syngnathid-specific losses in the family of fibroblast growth factors (FGFs), which likely involve reorganization of highly conserved gene regulatory networks in ways that have not previously been documented in natural populations. The resources presented here serve as important tools for future evolutionary studies of developmental processes in syngnathids and hold value for conservation of the extravagant seadragons and their relatives.

Seadragons are phenotypic outliers in an already exceptional clade of teleost fishes (family Syngnathidae) that also includes seahorses and pipefishes. For this reason, seadragons are often a colorful, flagship group in discussions of adaptation and evolutionary innovation. They show strikingly derived characters compared to their pipefish and seahorse relatives, including “leafy appendages,” extreme curvature of the spine (kyphosis and lordosis), elongated craniofacial bones, and large body size (Fig. 1) (1, 2). Substantial differences exist even among the three known extant species: Phycodurus eques (leafy seadragon), Phyllopteryx taeniolatus (weedy, or common, seadragon), and the recently described Phyllopteryx dewysea (ruby seadragon) (2).Open in a separate windowFig. 1.The anatomy of the weedy seadragon includes remarkably elongated facial features terminating in toothless, upturned jaws, an unusual hyoid apparatus specialized for suction feeding, a bony exoskeleton with elaborate spines that support fleshy leaves, and a sinusoidal spine of ribless vertebrae that vary in shape and size. (A) Lateral view of the skeleton of P. taeniolatus reconstructed by X-ray microscopy. (B) Detail of the head (the ceratohyal, c, and urohyal, u, of the hyoid apparatus are noted). (C) Detail of the pectoral region (lateral view) showing a dorsal, unpaired “leafy” appendage support surrounded by other dermal plates with much shorter spines. (D) Optical cross-section of the tail through a pair of leafy appendage spines. (E) Optical cross-section through the same appendages as in D but with a contrast agent that reveals the fleshy leaves, as denoted by (l). (F) Lateral view shows keystone-shaped vertebrae at curvatures—both kyphosis and lordosis—of the spine. (G) Ventral view of the ribless abdominal vertebrae. (H) Lateral detail of the specialized vertebrae beneath the propulsive dorsal fin.In addition to being a focus for evolutionary studies, seadragons are of significant cultural and conservation interest (3–5). Presumed adaptations for crypsis, including the leafy appendages, unique body plan, and elaborate skin coloration, contribute to the status of seadragons as distinguished and valued cultural symbols for the people of Australia, where seadragon species are endemic. Because seadragon distributions are specific to temperate Australian macroalgal reefs, and their population sizes are relatively small, seadragons are likely susceptible to negative human impacts, including global climate change. Furthermore, recent population genomic studies documenting significant population structure (6, 7) in these species are especially relevant to conservation decisions. The unique evolutionary innovations, cultural importance, and conservation challenges all elevate the need to better understand and conserve seadragon species.To improve our understanding of highly derived phenotypic traits and genomic features within seadragons, as well as those that are shared but derived among the Syngnathidae, we created annotated, chromosome-scale assemblies for a male leafy seadragon and a female weedy seadragon. In addition to the production of these resources, we carried out several comparative analyses among five syngnathid and many other teleost genomes to determine changes in genome organization and content, including a detailed analysis of key gene families and regulatory elements that may be involved in the development of syngnathid innovations. Lastly, we performed high-resolution three-dimensional (3D) X-ray microscope scans of an adult male weedy seadragon to more precisely view seadragon innovations.Our work reveals several seadragon-specific genomic features, including divergent conserved noncoding elements (CNEs) near key developmental genes, a unique microRNA gene repertoire, and expanded gene families related to immunity and vesicular trafficking. We also found that the seadragon genomes are highly repetitive for their sizes, with unique repeat abundance distributions. Because the seadragon lineage occupies a region of the syngnathid phylogeny that is relatively basal to most of the species’ diversity, we leveraged their phylogenetic position to identify several genomic synampomorphies of the family. These genomic features include the striking loss of several highly conserved fibroblast growth factor (FGF) genes, expansions and contractions of gene families related to immunity and potentially male pregnancy, and syngnathid-specific transposable element (TE) expansion.With these genome models and rich accompanying data, we add to the existing collection of high-quality genomic tools and insights for several syngnathid groups, including genera Syngnathus (8, 9), Hippocampus (10, 11), Microphis (12), and most recently (published as of the writing of this paper) Phyllopteryx and Syngnathoides (13). Such tools are useful in illuminating the evolution and development of puzzling syngnathid novelties such as male pregnancy and leaf-like appendages. These genomic resources will also support ongoing efforts to understand and conserve sensitive syngnathid populations, including phylogenetic umbrella species like the seadragons.     

Structures of two core subunits of the bacterial type IV secretion system, VirB8 from Brucella suis and ComB10 from Helicobacter pylori

Type IV secretion systems (T4SSs) are commonly used secretion machineries in Gram-negative bacteria. They are used in the infection of human, animal, or plant cells and the propagation of antibiotic resistance. The T4SS apparatus spans both membranes of the bacterium and generally is composed of 12 proteins, named VirB1-11 and VirD4 after proteins of the canonical Agrobacterium tumefaciens T4SS. The periplasmic core complex of VirB8/VirB10 structurally and functionally links the cytoplasmic NTPases of the system with its outer membrane and pilus components. Here we present crystal structures of VirB8 of Brucella suis, the causative agent of brucellosis, and ComB10, a VirB10 homolog of Helicobacter pylori, the causative agent of gastric ulcers. The structures of VirB8 and ComB10 resemble known folds, albeit with novel secondary-structure modifications unique to and conserved within their respective families. Both proteins crystallized as dimers, providing detailed predictions about their self associations. These structures make a substantial contribution to the repertoire of T4SS component structures and will serve as springboards for future functional and protein-protein interaction studies by using knowledge-based site-directed and deletion mutagenesis.     

Broad disorder and the allosteric mechanism of myosin II regulation by phosphorylation

Double electron electron resonance EPR methods was used to measure the effects of the allosteric modulators, phosphorylation, and ATP, on the distances and distance distributions between the two regulatory light chain of myosin (RLC). Three different states of smooth muscle myosin (SMM) were studied: monomers, the short-tailed subfragment heavy meromyosin, and SMM filaments. We reconstituted myosin with nine single cysteine spin-labeled RLC. For all mutants we found a broad distribution of distances that could not be explained by spin-label rotamer diversity. For SMM and heavy meromyosin, several sites showed two heterogeneous populations in the unphosphorylated samples, whereas only one was observed after phosphorylation. The data were consistent with the presence of two coexisting heterogeneous populations of structures in the unphosphorylated samples. The two populations were attributed to an on and off state by comparing data from unphosphorylated and phosphorylated samples. Models of these two states were generated using a rigid body docking approach derived from EM [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366] (PNAS, 2001, 98:4361-4366), but our data revealed a new feature of the off-state, which is heterogeneity in the orientation of the two RLC. Our average off-state structure was very similar to the Wendt model reveal a new feature of the off state, which is heterogeneity in the orientations of the two RLC. As found previously in the EM study, our on-state structure was completely different from the off-state structure. The heads are splayed out and there is even more heterogeneity in the orientations of the two RLC.     

From the Cover: Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization

As an economic crop, pepper satisfies people’s spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum). We estimate that the pepper genome expanded ∼0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of ∼81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs.Pepper (Capsicum) is an economically important genus of the Solanaceae family, which also includes tomato and potato. The genus includes at least 32 species native to tropical America (1), of which C. annuum L., C. baccatum L., C. chinense Jacq., C. frutescens L., and C. pubescens (Ruiz & Pavon) were domesticated as far back as 6000 B.C. by Native Americans (2). Peppers have a wide diversity of fruit shape, size, and color. Pungent peppers are used as spices, and sweet peppers are used as vegetables. After the return of Columbus from America in 1492 and subsequent voyages of exploration, peppers spread around the world because of adaptation to different agroclimatic regions and rapid adoption of pepper in different cultures as food, medicine, and ornamentals (3, 4). Pepper global production in 2011 reached 34.6 million tons fresh fruit and 3.5 million tons dried pods harvested in 3.9 million hectares (www.fao.org). Despite the growing commercial importance of pepper, the molecular mechanisms that modulate fruit size, shape, and yield are mostly unknown.Since the 1990s, genetic diversity and allelic shifts among cultivars, domesticated landraces, and wild accessions have been partially explored using restricted sets of anonymous or neutral molecular markers (5–9) and annotated DNA sequences (10). These studies reported that the genetic variability among sweet and large-fruited C. annuum cultivars was very restricted and suggested that changes in the allelic frequencies and a subsequent loss of diversity during the transition from wild to cultivated populations occurred even in areas of species cohabitation. The relatively low levels of genetic diversity in the primary gene pool have constrained pepper genetic improvement. Another primary reason for limited applied and basic research in pepper has been lack of a reference genome sequence of ∼3.3 Gb (11). Recent work comparing two members of the Solanaceae family (pepper and tomato) has begun to shed light on the processes that influence the dynamics of genome size in angiosperms (12, 13).To contribute to the understanding of pepper biology and evolution and accelerate agricultural applications, we generated and analyzed two reference genome sequences of cultivated Zunla-1 and wild Chiltepin (2n = 2x = 24). The two pepper genomes together with 20 resequencing accessions, including 3 accessions that are classified as semiwild/wild, provide a better understanding of the evolution, domestication, and divergence of various pepper species and ultimately, will enhance future genetic improvement of this important worldwide crop.     

Molecular basis for Nup37 and ELY5/ELYS recruitment to the nuclear pore complex

Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs), enormous assemblies composed of multiple copies of ∼30 different proteins called nucleoporins. To unravel the basic scaffold underlying the NPC, we have characterized the species-specific scaffold nucleoporin Nup37 and ELY5/ELYS. Both proteins integrate directly via Nup120/160 into the universally conserved heptameric Y-complex, the critical unit for the assembly and functionality of the NPC. We present the crystal structure of Schizosaccharomyces pombe Nup37 in complex with Nup120, a 174-kDa subassembly that forms one of the two short arms of the Y-complex. Nup37 binds near the bend of the L-shaped Nup120 protein, potentially stabilizing the relative orientation of its two domains. By means of reconstitution assays, we pinpoint residues crucial for this interaction. In vivo and in vitro results show that ELY5 binds near an interface of the Nup120–Nup37 complex. Complementary biochemical and cell biological data refine and consolidate the interactions of Nup120 within the current Y-model. Finally, we propose an orientation of the Y-complex relative to the pore membrane, consistent with the lattice model.     

Differentiation of the maize subgenomes by genome dominance and both ancient and ongoing gene loss

Ancient tetraploidies are found throughout the eukaryotes. After duplication, one copy of each duplicate gene pair tends to be lost (fractionate). For all studied tetraploidies, the loss of duplicated genes, known as homeologs, homoeologs, ohnologs, or syntenic paralogs, is uneven between duplicate regions. In maize, a species that experienced a tetraploidy 5-12 million years ago, we show that in addition to uneven ancient gene loss, the two complete genomes contained within maize are differentiated by ongoing fractionation among diverse inbreds as well as by a pattern of overexpression of genes from the genome that has experienced less gene loss. These expression differences are consistent over a range of experiments quantifying RNA abundance in different tissues. We propose that the universal bias in gene loss between the genomes of this ancient tetraploid, and perhaps all tetraploids, is the result of selection against loss of the gene responsible for the majority of total expression for a duplicate gene pair. Although the tetraploidy of maize is ancient, biased gene loss and expression continue today and explain, at least in part, the remarkable genetic diversity found among modern maize cultivars.