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Disease susceptibility and resistance are important factors for the conservation of endangered species, including elephants. We analyzed pathology data from 26 zoos and report that Asian elephants have increased neoplasia and malignancy prevalence compared with African bush elephants. This is consistent with observed higher susceptibility to tuberculosis and elephant endotheliotropic herpesvirus (EEHV) in Asian elephants. To investigate genetic mechanisms underlying disease resistance, including differential responses between species, among other elephant traits, we sequenced multiple elephant genomes. We report a draft assembly for an Asian elephant, and defined 862 and 1,017 conserved potential regulatory elements in Asian and African bush elephants, respectively. In the genomes of both elephant species, conserved elements were significantly enriched with genes differentially expressed between the species. In Asian elephants, these putative regulatory regions were involved in immunity pathways including tumor-necrosis factor, which plays an important role in EEHV response. Genomic sequences of African bush, forest, and Asian elephant genomes revealed extensive sequence conservation at TP53 retrogene loci across three species, which may be related to TP53 functionality in elephant cancer resistance. Positive selection scans revealed outlier genes related to additional elephant traits. Our study suggests that gene regulation plays an important role in the differential inflammatory response of Asian and African elephants, leading to increased infectious disease and cancer susceptibility in Asian elephants. These genomic discoveries can inform future functional and translational studies aimed at identifying effective treatment approaches for ill elephants, which may improve conservation.

Disease susceptibility and resistance comprise important factors in conservation, particularly in elephants. To determine genetic mechanisms underlying disease resistance and other unique elephant traits, we estimated 862 and 1,017 potential regulatory elements in Asian and African elephants, respectively. These elements are significantly enriched in both species with differentially expressed genes involved in immunity pathways, including tumor-necrosis factor which plays a role in the response to elephant endotheliotropic herpesvirus (EEHV). Population genomics analyses indicate that amplifiedTP53retrogenes are maintained by purifying selection and may contribute to cancer resistance in elephants, including less malignancies in African vs. Asian elephants. Positive selection scans across elephant genomes revealed genes that may control iconic elephant traits such as tusk development, memory, and somatic maintenance. Our study supports the hypothesis that interspecies variation in gene regulation contributes to differential inflammatory responses leading to increased infectious disease and cancer susceptibility in Asian versus African elephants. Genomics can inform functional immunological studies which may improve both conservation for elephants and human therapies.

Transferable DNA markers are essential for breeding and genetics. Grapevine (Vitis) breeders utilize disease resistance alleles from congeneric species ~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linked-read de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those ~20 million years divergent., Trait introgression requires universal markers, but cross-species transferability of current SNP markers can be as low as 2%. Here, the authors use an AmpSeq haplotype strategy targeting the collinear core genome for marker development and show transferability increases to 91.4% in the Vitis genus.

Motivation The accurate interpretation of genetic variants is critical for characterizing genotype–phenotype associations. Because the effects of genetic variants can depend strongly on their local genomic context, accurate genome annotations are essential. Furthermore, as some variants have the potential to disrupt or alter gene structure, variant interpretation efforts stand to gain from the use of individualized annotations that account for differences in gene structure between individuals or strains. Results We describe a suite of software tools for identifying possible functional changes in gene structure that may result from sequence variants. ACE (‘Assessing Changes to Exons’) converts phased genotype calls to a collection of explicit haplotype sequences, maps transcript annotations onto them, detects gene-structure changes and their possible repercussions, and identifies several classes of possible loss of function. Novel transcripts predicted by ACE are commonly supported by spliced RNA-seq reads, and can be used to improve read alignment and transcript quantification when an individual-specific genome sequence is available. Using publicly available RNA-seq data, we show that ACE predictions confirm earlier results regarding the quantitative effects of nonsense-mediated decay, and we show that predicted loss-of-function events are highly concordant with patterns of intolerance to mutations across the human population. ACE can be readily applied to diverse species including animals and plants, making it a broadly useful tool for use in eukaryotic population-based resequencing projects, particularly for assessing the joint impact of all variants at a locus. Availability and Implementation ACE is written in open-source C ++ and Perl and is available from geneprediction.org/ACE Supplementary information Supplementary information is available at Bioinformatics online.

Motivation Genetic variation that disrupts gene function by altering gene splicing between individuals can substantially influence traits and disease. In those cases, accurately predicting the effects of genetic variation on splicing can be highly valuable for investigating the mechanisms underlying those traits and diseases. While methods have been developed to generate high quality computational predictions of gene structures in reference genomes, the same methods perform poorly when used to predict the potentially deleterious effects of genetic changes that alter gene splicing between individuals. Underlying that discrepancy in predictive ability are the common assumptions by reference gene finding algorithms that genes are conserved, well-formed and produce functional proteins. Results We describe a probabilistic approach for predicting recent changes to gene structure that may or may not conserve function. The model is applicable to both coding and non-coding genes, and can be trained on existing gene annotations without requiring curated examples of aberrant splicing. We apply this model to the problem of predicting altered splicing patterns in the genomes of individual humans, and we demonstrate that performing gene-structure prediction without relying on conserved coding features is feasible. The model predicts an unexpected abundance of variants that create de novo splice sites, an observation supported by both simulations and empirical data from RNA-seq experiments. While these de novo splice variants are commonly misinterpreted by other tools as coding or non-coding variants of little or no effect, we find that in some cases they can have large effects on splicing activity and protein products and we propose that they may commonly act as cryptic factors in disease. Availability and implementation The software is available from geneprediction.org/SGRF. Supplementary information Supplementary information is available at Bioinformatics online.

The evolution of transcriptional regulatory mechanisms is central to how stress response and tolerance differ between species. However, it remains largely unknown how divergence in cis-regulatory sites and, subsequently, transcription factor (TF) binding specificity contribute to stress-responsive expression divergence, particularly between wild and domesticated species. By profiling wound-responsive gene transcriptomes in wild Solanum pennellii and domesticated S. lycopersicum, we found extensive wound response divergence and identified 493 S. lycopersicum and 278 S. pennellii putative cis-regulatory elements (pCREs) that were predictive of wound-responsive gene expression. Only 24-52% of these wound response pCREs (depending on wound response patterns) were consistently enriched in the putative promoter regions of wound-responsive genes across species. In addition, between these two species, their differences in pCRE site sequences were significantly and positively correlated with differences in wound-responsive gene expression. Furthermore, ∼11-39% of pCREs were specific to only one of the species and likely bound by TFs from different families. These findings indicate substantial regulatory divergence in these two plant species that diverged ∼3-7 million years ago. Our study provides insights into the mechanistic basis of how the transcriptional response to wounding is regulated and, importantly, the contribution of cis-regulatory components to variation in wound-responsive gene expression between a wild and a domesticated plant species.

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

Acquisition and storage of Latimeria chalumnae samples was supported by grants from the African Coelacanth Ecosystem Programme of the South African National Department of Science and Technology. Generation of the Latimeria chalumnae and Protopterus annectens sequences by the Broad Institute of the Massachusetts Institute of Technology (MIT) and Harvard University was supported by grants from the National Human Genome Research Institute (NHGRI). K.L.T. is the recipient of a EURYI award from the European Science Foundation. We would also like to thank the Genomics Sequencing Platform of the Broad Institute for sequencing the L. chalumnae genome and L. chalumnae and P. annectens transcriptomes, S. Ahamada, R. Stobbs and the Association pour le Protection de Gombesa (APG) for their help in obtaining coelacanth samples, Y. Zhao for the use of data from Rana chensinensis, and L. Gaffney, C. Hamilton and J. Westlund for assistance with figure preparation. 10; International audience; The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

The large size and relative complexity of many plant genomes make creation, quality control, and dissemination of high-quality gene structure annotations challenging. In response, we have developed MAKER-P, a fast and easy-to-use genome annotation engine for plants. Here, we report the use of MAKER-P to update and revise the maize (Zea mays) B73 RefGen_v3 annotation build (5b+) in less than 3 h using the iPlant Cyberinfrastructure. MAKER-P identified and annotated 4,466 additional, well-supported protein-coding genes not present in the 5b+ annotation build, added additional untranslated regions to 1,393 5b+ gene models, identified 2,647 5b+ gene models that lack any supporting evidence (despite the use of large and diverse evidence data sets), identified 104,215 pseudogene fragments, and created an additional 2,522 noncoding gene annotations. We also describe a method for de novo training of MAKER-P for the annotation of newly sequenced grass genomes. Collectively, these results lead to the 6a maize genome annotation and demonstrate the utility of MAKER-P for rapid annotation, management, and quality control of grasses and other difficult-to-annotate plant genomes.

Gramene (http://www.gramene.org) is a knowledgebase for comparative functional analysis in major crops and model plant species. The current release, #54, includes over 1.7 million genes from 44 reference genomes, most of which were organized into 62,367 gene families through orthologous and paralogous gene classification, whole-genome alignments, and synteny. Additional gene annotations include ontology-based protein structure and function; genetic, epigenetic, and phenotypic diversity; and pathway associations. Gramene's Plant Reactome provides a knowledgebase of cellular-level plant pathway networks. Specifically, it uses curated rice reference pathways to derive pathway projections for an additional 66 species based on gene orthology, and facilitates display of gene expression, gene–gene interactions, and user-defined omics data in the context of these pathways. As a community portal, Gramene integrates best-of-class software and infrastructure components including the Ensembl genome browser, Reactome pathway browser, and Expression Atlas widgets, and undergoes periodic data and software upgrades. Via powerful, intuitive search interfaces, users can easily query across various portals and interactively analyze search results by clicking on diverse features such as genomic context, highly augmented gene trees, gene expression anatomograms, associated pathways, and external informatics resources. All data in Gramene are accessible through both visual and programmatic interfaces.

An improved reference genome for maize, using single-molecule sequencing and high-resolution optical mapping, enables characterization of structural variation and repetitive regions, and identifies lineage expansions of transposable elements that are unique to maize. Supplementary information The online version of this article (doi:10.1038/nature22971) contains supplementary material, which is available to authorized users., A better map of the maize genome The maize genome was initially reported in 2009 but with some accuracy limitations. Doreen Ware and colleagues report a new reference genome for maize using single-molecule sequencing and high-resolution optical mapping. The technique shows improvements in the gene space including resolution of gaps and misassemblies and correction of order and orientation of genes. The authors characterize structural variation and repetitive regions, and identify transposable element lineage expansions unique to maize. Supplementary information The online version of this article (doi:10.1038/nature22971) contains supplementary material, which is available to authorized users., Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation1. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions2. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome3, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing4. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes. Supplementary information The online version of this article (doi:10.1038/nature22971) contains supplementary material, which is available to authorized users.

BackgroundThe evolution ofcis-andtrans-regulatory components of transcription is central to how stress response and tolerance differ across species. However, it remains largely unknown how divergence in TF binding specificity andcis-regulatory sites contribute to the divergence of stress-responsive gene expression between wild and domesticated species.ResultsUsing tomato as model, we analyzed the transcriptional profile of wound-responsive genes in wildSolanum pennelliiand domesticatedS. lycopersicum. We found that extensive expression divergence of wound-responsive genes is associated with speciation. To assess the degree of trans-regulatory divergence between these two species, 342 and 267 putativecis-regulatory elements (pCREs) inS. lycopersicumandS. pennellii, respectively, were identified that were predictive of wound-induced gene expression. We found that 35-66% of pCREs were conserved across species, suggesting that the remaining proportion (34-65%) of pCREs are species specific. This finding indicates a substantially higher degree of trans-regulatory divergence between these two plant species, which diverged ∼3-7 million years ago, compared to that observed in mouse and human, which diverged ∼100 million years ago. In addition, differences in pCRE sites were significantly associated with differences in wound-responsive gene expression between wild and domesticated tomato orthologs, suggesting the presence of substantialcis-regulatory divergence.ConclusionsOur study provides new insights into the mechanistic basis of how the transcriptional response to wounding is regulated and, importantly, the contribution ofcis-andtrans-regulatory components to variation in wound-responsive gene expression during species domestication.

Southern right whales (SRWs, Eubalena australis) are polymorphic for an X-linked pigmentation pattern known as grey morphism. Most SRWs have completely black skin with white patches on their bellies and occasionally on their backs; these patches remain white as the whale ages. Grey morphs (previously referred to as partial albinos) appear mostly white at birth, with a splattering of rounded black marks; but as the whales age, the white skin gradually changes to a brownish grey color. The cellular and developmental bases of grey morphism are not understood. Here we describe cellular and ultrastructural features of grey-morph skin in relation to that of normal, wild-type skin. Melanocytes were identified histologically and counted, and melanosomes were measured using transmission electron microscopy. Grey-morph skin had fewer melanocytes when compared to wild-type skin, suggesting reduced melanocyte survival, migration, or proliferation in these whales. Grey-morph melanocytes had smaller melanosomes relative to wild-type skin, normal transport of melanosomes to surrounding keratinocytes, and normal localization of melanin granules above the keratinocyte nuclei. These findings indicate that SRW grey-morph pigmentation patterns are caused by reduced numbers of melanocytes in the skin, as well as by reduced amounts of melanin production and/or reduced sizes of mature melanosomes. Grey morphism is distinct from piebaldism and albinism found in other species, which are genetic pigmentation conditions resulting from the local absence of melanocytes, or the inability to synthesize melanin, respectively.This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Fil: Eroh, Guy D.. Huntsman Cancer Institute; Estados Unidos. University of Georgia; Estados Unidos Fil: Clayton, Fred C.. University of Utah; Estados Unidos Fil: Florell, Scott R.. University of Utah; Estados Unidos Fil: Cassidy, Pamela B.. Huntsman Cancer Institute; Estados Unidos. Oregon Health & Science University; Estados Unidos Fil: Chirife, Andrea. Instituto de Conservación de Ballenas. Programa de Monitoreo Sanitario de la Ballena Franca Austral; Argentina Fil: Marón, Carina Flavia. University of Utah; Estados Unidos. Instituto de Conservación de Ballenas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Valenzuela, Luciano Oscar. University of Utah; Estados Unidos. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Sociales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Campbell, Michael S.. University of Utah; Estados Unidos. Cold Spring Harbor Laboratory; Estados Unidos Fil: Seger, Jon. University of Utah; Estados Unidos Fil: Rowntree, Victoria J.. University of Utah; Estados Unidos. Instituto de Conservación de Ballenas. Programa de Monitoreo Sanitario de la Ballena Franca Austral; Argentina. Ocean Alliance; Estados Unidos Fil: Leachman, Sancy A.. Oregon Health & Science University; Estados Unidos. Huntsman Cancer Institute; Estados Unidos

Maternal tobacco smoke (MTS) predisposes human and rat offspring to visceral obesity in early adulthood. Glucocorticoid excess also causes visceral obesity. We hypothesized that in utero MTS would increase visceral adiposity and alter the glucocorticoid pathway in young adult rats. We developed a novel model of in utero MTS exposure in pregnant rats by exposing them to cigarette smoke from E11.5 to term. Neonatal rats were cross-fostered to control dams and weaned to standard rat chow through young adulthood (postnatal day 60). We demonstrated increased visceral adiposity (193%)*, increased visceral adipose 11-β hydroxysteroid dehydrogenase 1 mRNA (204%)*, increased serum corticosterone (147%)*, and no change in glucocorticoid receptor protein in adult male MTS rat offspring. Female rats exposed to MTS in utero demonstrated no change in visceral or subcutaneous adiposity, decreased serum corticosterone (60%)*, and decreased adipose glucocorticoid receptor protein (66%)*. *P < 0.05. We conclude that in utero MTS exposure increased visceral adiposity and altered in the glucocorticoid pathway in a sex-specific manner. We speculate that in utero MTS exposure programs adipose dysfunction in adult male rat offspring via alteration in the glucocorticoid pathway.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate elucidation of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here, we report the assembly and annotation of maize, a genetic and agricultural model species, using Single Molecule Real-Time (SMRT) sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and significant improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed over 130,000 intact transposable elements (TEs), allowing us to identify TE lineage expansions unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by SMRT sequencing. In addition, comparative optical mapping of two other inbreds revealed a prevalence of deletions in the low gene density region and maize lineage-specific genes.

We have optimized and extended the widely used annotation engine MAKER in order to better support plant genome annotation efforts. New features include better parallelization for large repeat-rich plant genomes, noncoding RNA annotation capabilities, and support for pseudogene identification. We have benchmarked the resulting software tool kit, MAKER-P, using the Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) genomes. Here, we demonstrate the ability of the MAKER-P tool kit to automatically update, extend, and revise the Arabidopsis annotations in light of newly available data and to annotate pseudogenes and noncoding RNAs absent from The Arabidopsis Informatics Resource 10 build. Our results demonstrate that MAKER-P can be used to manage and improve the annotations of even Arabidopsis, perhaps the best-annotated plant genome. We have also installed and benchmarked MAKER-P on the Texas Advanced Computing Center. We show that this public resource can de novo annotate the entire Arabidopsis and maize genomes in less than 3 h and produce annotations of comparable quality to those of the current The Arabidopsis Information Resource 10 and maize V2 annotation builds.

Coo Coo Charles Darwin was fascinated by the domestic rock pigeon and used this dramatic example of diversity within a species to communicate his ideas about natural selection. Many derived traits in domestic pigeons converge on ecologically and evolutionarily relevant traits in wild species. Shapiro et al. (p. 1063 , published online 31 January; see the cover) sequenced the genome of the domestic rock pigeon ( Columba livia ), along with those of 36 breeds and two feral accessions and its sister species, the hill pigeon ( C. rupestris ). The results reveal the underlying genetics of the head crest and suggest that all crested breeds may have originated from a single mutational event.

Intrauterine growth restriction (IUGR) predisposes to obesity and adipose dysfunction. We previously demonstrated IUGR-induced increased visceral adipose deposition and dysregulated expression of peroxisome proliferator activated receptor-γ2 (PPARγ2) in male adolescent rats, prior to the onset of obesity. In other studies, activation of PPARγincreases subcutaneous adiponectin expression and normalizes visceral adipose deposition. We hypothesized that maternal supplementation with docosahexaenoic acid (DHA), a PPARγagonist, would normalize IUGR adipose deposition in association with increased PPARγ, adiponectin, and adiponectin receptor expression in subcutaneous adipose. To test these hypotheses, we used a well-characterized model of uteroplacental-insufficiency-(UPI-) induced IUGR in the rat with maternal DHA supplementation. Our primary findings were that maternal DHA supplementation during rat pregnancy and lactation (1) normalizes IUGR-induced changes in adipose deposition and visceral PPARγexpression in male rats and (2) increases serum adiponectin, as well as adipose expression of adiponectin and adiponectin receptors in former IUGR rats. Our novel findings suggest that maternal DHA supplementation may normalize adipose dysfunction and promote adiponectin-induced improvements in metabolic function in IUGR.

Genome projects have evolved from large international undertakings to tractable endeavors for a single lab. Accurate genome annotation is critical for successful genomic, genetic, and molecular biology experiments. These annotations can be generated using a number of approaches and available software tools. This unit describes methods for genome annotation and a number of software tools commonly used in gene annotation.

Importance Evolutionary medicine may provide insights into human physiology and pathophysiology, including tumor biology. Objective To identify mechanisms for cancer resistance in elephants and compare cellular response to DNA damage among elephants, healthy human controls, and cancer-prone patients with Li-Fraumeni syndrome (LFS). Design, Setting, and Participants A comprehensive survey of necropsy data was performed across 36 mammalian species to validate cancer resistance in large and long-lived organisms, including elephants (n = 644). The African and Asian elephant genomes were analyzed for potential mechanisms of cancer resistance. Peripheral blood lymphocytes from elephants, healthy human controls, and patients with LFS were tested in vitro in the laboratory for DNA damage response. The study included African and Asian elephants (n = 8), patients with LFS (n = 10), and age-matched human controls (n = 11). Human samples were collected at the University of Utah between June 2014 and July 2015. Exposures Ionizing radiation and doxorubicin. Main Outcomes and Measures Cancer mortality across species was calculated and compared by body size and life span. The elephant genome was investigated for alterations in cancer-related genes. DNA repair and apoptosis were compared in elephant vs human peripheral blood lymphocytes. Results Across mammals, cancer mortality did not increase with body size and/or maximum life span (eg, for rock hyrax, 1% [95% CI, 0%-5%]; African wild dog, 8% [95% CI, 0%-16%]; lion, 2% [95% CI, 0%-7%]). Despite their large body size and long life span, elephants remain cancer resistant, with an estimated cancer mortality of 4.81% (95% CI, 3.14%-6.49%), compared with humans, who have 11% to 25% cancer mortality. While humans have 1 copy (2 alleles) ofTP53, African elephants have at least 20 copies (40 alleles), including 19 retrogenes (38 alleles) with evidence of transcriptional activity measured by reverse transcription polymerase chain reaction. In response to DNA damage, elephant lymphocytes underwent p53-mediated apoptosis at higher rates than human lymphocytes proportional toTP53status (ionizing radiation exposure: patients with LFS, 2.71% [95% CI, 1.93%-3.48%] vs human controls, 7.17% [95% CI, 5.91%-8.44%] vs elephants, 14.64% [95% CI, 10.91%-18.37%];P Conclusions and Relevance Compared with other mammalian species, elephants appeared to have a lower-than-expected rate of cancer, potentially related to multiple copies ofTP53. Compared with human cells, elephant cells demonstrated increased apoptotic response following DNA damage. These findings, if replicated, could represent an evolutionary-based approach for understanding mechanisms related to cancer suppression.

This unit describes how to use the genome annotation and curation tools MAKER and MAKER-P to annotate protein coding and non-coding RNA genes in newly assembled genomes, update/combine legacy annotations in light of new evidence, add quality metrics to annotations from other pipelines, and map existing annotations to a new assembly. MAKER and MAKER-P can rapidly annotate genomes of any size, and scale to match available computational resources.

We present the de novo draft genome sequence for a vertebrate mammalian herbivore, the desert woodrat (Neotoma lepida). This species is of ecological and evolutionary interest with respect to ingestion, microbial detoxification and hepatic metabolism of toxic plant secondary compounds from the highly toxic creosote bush (Larrea tridentata) and the juniper shrub (Juniperus monosperma). The draft genome sequence and annotation have been deposited at GenBank under the accession LZPO01000000.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Carbone, Lucia et al.-- This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported licence., Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.

Intrauterine growth restriction (IUGR) programs adult disease, including obesity and insulin resistance. Our group previously demonstrated that IUGR dysregulates adipose deposition in male, but not female, weanling rats. Dysregulated adipose deposition is often accompanied by the release of proinflammatory signaling molecules, such as tumor necrosis factor alpha (TNFα). TNFαcontributes to adipocyte inflammation and impaired insulin signaling. TNFαhas also been implicated in the activation of the unfolded protein response (UPR), which impairs insulin signaling. We hypothesized that, in male rat pups, IUGR would increase TNFα, TNFR1, and components of the UPR (Hspa5, ATF6, p-eIF2α, and Ddit3) prior to the onset of obesity. We further hypothesized that impaired glucose tolerance would occur after the onset of adipose dysfunction in male IUGR rats. To test this hypothesis, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. Our primary findings are that, in male rats, IUGR (1) increased circulating and adipose TNFα, (2) increased mRNA levels of UPR components as well as p-eIF2a, and (3) impaired glucose tolerance after observed TNFαincreased and after UPR activation. We speculate that programmed dysregulation of TNFαand UPR contributed to the development of glucose intolerance in male IUGR rats.

The spindle checkpoint prevents errors in mitosis. Cells respond to the presence of kinetochores that are improperly attached to the mitotic spindle by delaying anaphase onset. Evidence suggests that phosphorylations recognized by the 3F3/2 anti-phosphoepitope antibody may be involved in the kinetochore signaling of the spindle checkpoint. Mitotic cells lysed in detergent in the absence of phosphatase inhibitors rapidly lose expression of the 3F3/2 phosphoepitope. However, when ATP is added to lysed and rinsed mitotic cytoskeletons, kinetochores become rephosphorylated by an endogenous, bound kinase. Kinetochore rephosphorylation in vitro produced the same differential phosphorylation seen in appropriately fixed living cells. In chromosomes not yet aligned at the metaphase plate, kinetochores undergo rapid rephosphorylation, while those of fully congressed chromosomes are under-phosphorylated. However, latent 3F3/2 kinase activity is retained at kinetochores of cells at all stages of mitosis including anaphase. This latent activity is revealed when rephosphorylation reactions are carried out for extended times. The endogenous, kinetochore-bound kinase can be chemically inactivated. Remarkably, a soluble kinase activity extracted from mitotic cells also caused differential rephosphorylation of kinetochores whose endogenous kinase had been chemically inactivated. We suggest that, in vivo, microtubule attachment alters the kinetochore 3F3/2 phosphoprotein, causing it to resist phosphorylation. This kinetochore modification is retained after cell lysis, producing a "memory" of the in vivo phosphorylation state.

ImagePlane is a modular pipeline for automated, high-throughput image analysis and information extraction. Designed to support planarian research, ImagePlane offers a self-parameterizing adaptive thresholding algorithm; an algorithm that can automatically segment animals into anterior–posterior/left–right quadrants for automated identification of region-specific differences in gene and protein expression; and a novel algorithm for quantification of morphology of animals, independent of their orientations and sizes. ImagePlane also provides methods for automatic report generation, and its outputs can be easily imported into third-party tools such as R and Excel. Here we demonstrate the pipeline's utility for identification of genes involved in stem cell proliferation in the planarian Schmidtea mediterranea. Although designed to support planarian studies, ImagePlane will prove useful for cell-based studies as well.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus., Author Summary Algae are a highly diverse group of organisms that have become the focus of renewed interest due to their potential for producing biofuel feedstocks, nutraceuticals, and biomaterials. Their high photosynthetic yields and ability to grow in areas unsuitable for agriculture provide a potential sustainable alternative to using traditional agricultural crops for biofuels. Because none of the algae currently in use have a history of domestication, and bioengineering of algae is still in its infancy, there is a need to develop algal strains adapted to cultivation for industrial large-scale production of desired compounds. Model organisms ranging from mice to baker's yeast have been instrumental in providing insights into fundamental biological structures and functions. The algal field needs versatile models to develop a fundamental understanding of photosynthetic production of biomass and valuable compounds in unicellular, marine, oleaginous algal species. To contribute to the development of such an algal model system for basic discovery, we sequenced the genome and two sets of transcriptomes of N. oceanica CCMP1779, assembled the genomic sequence, identified putative genes, and began to interpret the function of selected genes. This species was chosen because it is readily transformable with foreign DNA and grows well in culture.

The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation. Using video observation of migrating human neutrophils simultaneously illuminated for fluorescence and phase-contrast microscopy, we analyzed nuclear movements with respect to cell shape changes. The number of nuclear lobes and their relative size remained constant during observation (up to 1 h). The thin connecting segments between the lobes elongated and attenuated extensively but never separated. Electron microscopic analysis of neutrophil nuclei revealed no specialized nuclear or cytoplasmic structures in the vicinity of connecting segments. With fluorescence in situ hybridization of whole chromosome probes, we determined that chromosomes are randomly distributed among neutrophil nuclear lobes. HL60 cells are a human myelocytic line that, with retinoic acid treatment, segment their nuclei and differentiate into neutrophil-like cells over several days. Using a rapidly responding variant line termed HL60/S4 (Cancer Res. 52, 949–954), we found that segmentation could be induced within 24 h. We tested the role of cytoskeletal elements in the process of nuclear segmentation. Neither the microtubule inhibitor nocodazole nor the microfilament inhibitor cytochalasin D prevented nuclear segmentation. Together, our studies suggest that nuclear lobes in neutrophils are relatively stable structures that are not generated by microtubule- or microfilament-dependent forces.

We have detected novel phosphotyrosine epitopes at the kinetochores of mitotic chromosomes in rat kangaroo PtK1 and mouse P388D1 tissue culture cells. Immunofluorescence labeling of detergent-resistant cytoskeletons reveals that these phosphotyrosine epitopes are tightly bound at the centrosomes and kinetochores of mitotic cells. These phosphoepitopes are found at the kinetochores during only prophase and prometaphase. Inclusion of a mixture of phosphatase inhibitors in the cell extraction procedure was necessary to preserve these previously undetected phosphotyrosine epitopes. The use of the phosphatase inhibitor mixture also improved the detection of the centrosome and kinetochore antigens recognized by the monoclonal antibody MPM-2. The MPM-2 antibody labels a subset of phosphothreonine-containing antigens found primarily during M phase. Ultrastructural immunolabeling studies indicated that both the phosphotyrosine and the MPM-2 phosphoepitopes were contained in both the outer and the inner dense plaques of the kinetochore. We developed large-scale chromosome isolation procedures designed to maintain chromosome protein phosphorylation. Immunoblot analysis revealed that the phosphotyrosine and MPM-2 antibodies recognized a number of chromosomal proteins, some of which were concentrated in the chromosome scaffold fraction prepared by nuclease digestion and salt extraction of whole chromosomes. The strictly regulated appearance of the phosphotyrosine and MPM-2 epitopes at the kinetochores of chromosomes during various stages of mitosis suggests that these phosphoepitopes may be involved in signal transduction pathways controlling kinetochore assembly and function during mitosis.

Nat. Genet. 48, 427–437 (2016); published online 7 March 2016; corrected after print 25 April 2016 As we intended, other researchers have been able to use the draft spotted gar genome sequence available from the Broad Institute website since December 2011, the assembly LepOcu1 publicly available from NCBI since 13 January 2012 under accession code GCA000242695.

Complications of intrauterine growth restriction (IUGR) include increased pulmonary morbidities and impaired alveolar development. Normal alveolar development depends upon elastin expression and processing, as well as the formation and deposition of elastic fibers. This is true of the human and rat. In this study, we hypothesized that uteroplacental insufficiency (UPI)-induced IUGR decreases mRNA levels of elastin and genes required for elastin fiber synthesis and assembly, at birth (prealveolarization) and postnatal day 7 (midalveolarization) in the rat. We further hypothesized that this would be accompanied by reduced elastic fiber deposition and increased static compliance at postnatal day 21 (mature lung). We used a well characterized rat model of IUGR to test these hypotheses. IUGR decreases mRNA transcript levels of genes essential for elastic fiber formation, including elastin, at birth and day 7. In the day 21 lung, IUGR decreases elastic fiber deposition and increases static lung compliance. We conclude that IUGR decreases mRNA transcript levels of elastic fiber synthesis genes, before and during alveolarization leading to a reduced elastic fiber density and increased static lung compliance in the mature lung. We speculate that the mechanism by which IUGR predisposes to pulmonary disease may be via decreased lung elastic fiber deposition.

Uteroplacental insufficiency (UPI) induced intrauterine growth restriction (IUGR) predisposes individuals to adult onset metabolic morbidities, including insulin resistance and cardiovascular disease. An underlying component of the development of these morbidities is adipose dysfunction; specifically a disproportionately abundant visceral adipose tissue. We hypothesize that IUGR will increase rats visceral adiposity and visceral expression of PPARgamma, a key regulator of adipogenesis. To test this hypothesis we employed a well described UPI induced IUGR rat model. Subcutaneous and visceral adipose levels were measured in adolescent control and IUGR rats using MRI. Expression of PPARgamma mRNA and protein, as well as PPARgamma target genes, was measured in neonatal, adolescent and adult rats. UPI induced IUGR increases the relative amount of visceral adipose tissue in male, but not female, adolescent rats in conjunction with an increase in PPARgamma2mRNA and protein in male visceral adipose. Importantly, these effects are seen prior to the onset of overt obesity. We conclude that increased PPARgamma2 expression in VAT of IUGR males is associated with increased visceral adiposity. We speculate that the increase in visceral adiposity may contribute to the metabolic morbidities experienced by this population.

Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. We examined the subcellular distribution of Cdc27 in greater detail in mammalian cells and found Cdc27 concentrated at spindle poles and on spindle microtubules as previously described, but also found Cdc27 at kinetochores and along chromosome arms. This localization was not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyzed the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody resulted in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis.

The kinetochore, a macromolecular complex located at the centromere of chromosomes, provides essential functions for accurate chromosome segregation1,2. Kinetochores contain checkpoint proteins that monitor attachments between the kinetochore and microtubules to ensure that cells do not exit mitosis in the presence of unaligned chromosomes3,4. Here we report that human CENP-I, a constitutive protein of the kinetochore that shares limited similarity with Mis6 of Schizosaccharomyces pombe, is required for the localization of CENP-F and the checkpoint proteins MAD1 and MAD2 to kinetochores. Depletion of CENP-I from kinetochores causes the cell cycle to delay in G2. Although monopolar chromosomes in CENP-I-depleted cells fail to establish bipolar connections, the cells are unable to arrest in mitosis. These cells are transiently delayed in mitosis in a MAD2-dependent manner, even though their kinetochores are depleted of MAD2. The delay is extended considerably when the number of unattached kinetochores is increased. This suggests that no single unattached kinetochore in CENP-I-depleted cells can arrest mitosis. The collective output from many unattached kinetochores is required to reach a threshold signal of 'wait for anaphase' to sustain a prolonged mitotic arrest.

The rad17 gene of Schizosaccharomyces pombe plays an important role as a checkpoint protein following DNA damage and during DNA replication. The human homologue of S. pombe rad17, Hrad17, was recently identified, but its function has not yet been established. Using the yeast two-hybrid system, we determined that HRad17 can interact with a nucleolar protein, NHP2L1. This interaction was also demonstrated biochemically, in human cells. Immunofluorescence studies revealed that HRad17 and NHP2L1 colocalize to the nucleolus, and immunogold labeling further resolved the location of NHP2L1 to the dense fibrillar component of the nucleolus. Interestingly, the localization of HRad17 in the nucleolus was altered in response to UV irradiation. These results provide some insight into the DNA damage and replication checkpoint mechanisms of HRad17.

The transition from metaphase to anaphase is regulated by a checkpoint system that prevents chromosome segregation in anaphase until all the chromosomes have aligned at the metaphase plate. We provide evidence indicating that a kinetochore phosphoepitope plays a role in this checkpoint pathway. The 3F3/2 monoclonal antibody recognizes a kinetochore phosphoepitope in mammalian cells that is expressed on chromosomes before their congression to the metaphase plate. Once chromosomes are aligned, expression is lost and cells enter anaphase shortly thereafter. When microinjected into prophase cells, the 3F3/2 antibody caused a concentration-dependent delay in the onset of anaphase. Injected antibody inhibited the normal dephosphorylation of the 3F3/2 phosphoepitope at kinetochores. Microinjection of the antibody eliminated the asymmetric expression of the phosphoepitope normally seen on sister kinetochores of chromosomes during their movement to the metaphase plate. Chromosome movement to the metaphase plate appeared unaffected in cells injected with the antibody suggesting that asymmetric expression of the phosphoepitope on sister kinetochores is not required for chromosome congression to the metaphase plate. In antibody-injected cells, the epitope remained expressed at kinetochores throughout the prolonged metaphase, but had disappeared by the onset of anaphase. When normal cells in metaphase, lacking the epitope at kinetochores, were treated with agents that perturb microtubules, the 3F3/2 phosphoepitope quickly reappeared at kinetochores. Immunoelectron microscopy revealed that the 3F3/2 epitope is concentrated in the middle electronlucent layer of the trilaminar kinetochore structure. We propose that the 3F3/2 kinetochore phosphoepitope is involved in detecting stable kinetochore-microtubule attachment or is a signaling component of the checkpoint pathway regulating the metaphase to anaphase transition.

To the Editor: —In reading the celebrated diary of Samuel Pepys I recently came across the first account, so far as I know, of the transfusion of blood, which I think will be of interest to the readers ofThe Journal. The following is an extract: "Nov. the 14th, 1666. Dr. Croone told me that at the meeting at Gresham College to-night (which, it seems, they now have every Wednesday again), there was a pretty experiment of the blood of one dog let out (till he died) into the body of another on one side, while all his own run out on the other side. The first died upon the place, and the other very well, and likely to do well. This did give occasion to many pretty wishes, as of the blood of a Quaker to be let into an Archbishop, and such like; but, as Dr. Croone says