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Several species in the oomycete genus Peronosclerospora cause downy mildew on maize and can result in significant yield losses in Asia. Biosurveillance of these pathogens is a high priority to prevent epidemics on maize in the US and consequent damage to the US economy. The unresolved taxonomy and dearth of molecular resources for Peronosclerospora spp. hinder these efforts. P. sorghi is a pathogen of sorghum and maize with a global distribution, for which limited diversity has been detected in the southern USA. We characterized the genome, transcriptome, and mitogenome of an isolate, representing the US pathotype 6. The highly homozygous genome was assembled using 10x Genomics linked reads and scaffolded using Hi-C into 13 chromosomes. The total assembled length was 319.6 Mb—larger than any other oomycete previously assembled. The mitogenome was 38 kb, similar in size to other oomycetes, although it had a unique gene order. Nearly 20,000 genes were annotated in the nuclear genome, more than described for other downy mildew causing oomycetes. The 13 chromosomes of P. sorghi were highly syntenic with the 17 chromosomes of Peronospora effusa with conserved centromeric regions and distinct chromosomal fusions. The increased assembly size and gene count of P. sorghi is due to extensive retrotransposition, resulting in putative pseudogenization. Ancestral genes had higher transcript abundance and were enriched for differential expression. This study provides foundational resources for analysis of Peronosclerospora and comparisons to other oomycete genera. Further genomic studies of global Peronosclerospora spp. will determine the suitability of the mitogenome, ancestral genes, and putative pseudogenes for marker development and taxonomic relationships.

STUDY QUESTION How does ectopic endometrial stromal cell (Ecto-ESC)-derived extracellular vesicular Legumain pseudogene 1 (EV-LGMNP1), a newly identified pseudogene of Legumain (LGMN), contribute to M2-phenotype macrophage polarization, and does it predict recurrence in patients with ovarian endometriosis (EMs)? SUMMARY ANSWER EV-LGMNP1, which is abundant in Ecto-ESCs and serum from ovarian EMs, can direct macrophages towards an M2 phenotype by upregulating LGMN expression and is a promising biomarker for predicting ovarian EMs recurrence. WHAT IS KNOWN ALREADY Extracellular vesicles (EVs) can mediate cell-to-cell crosstalk to promote disease progression via cargo molecule transport. Recently, LGMNP1, a newly identified pseudogene of LGMN, has been reported to promote cancer progression by upregulating LGMN. LGMN is a well-studied protein that can induce M2-like polarization. STUDY DESIGN, SIZE, DURATION An in vitro study was conducted with Ecto-ESCs isolated from ectopic endometrial samples, collected from two patients with ovarian EMs (diagnosed by laparoscopy and histological analysis). A clinical retrospective cohort study of 52 ovarian EMs patients and 21 controls with available preoperative serum samples was carried out (2013–2017). The follow-up period ended either at the time of recurrence or on 31 December 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS Ecto-ESC-derived EVs (EV/Ecto-ESCs) were characterized by nanoparticle tracking analysis, transmission electron microscopy and western blotting. EV internalization by THP-1 cells, which are the most widely used primary human macrophages model, was detected by fluorescence labelling. After EV treatment, THP-1 cell polarization was detected by quantitative real-time PCR (qRT-PCR) and western blot analyses of CD86 (M1-related marker) and CD206 (M2-related marker). LGMNP1 mRNA expression level in EVs from both primary ectopic endometrioc stromal cells and serum was examined using qRT-PCR. Additionally, the expression of LGMN, the downstream target gene of LGMNP1, in THP-1 cells was evaluated using qRT-PCR and western blotting. Kaplan–Meier and multivariate Cox regression analyses were applied to evaluate the independent predictive factors of EMs recurrence-free survival. A novel nomogram model based on serum EV-LGMNP1 was then formulated to predict EMs recurrence. MAIN RESULTS AND THE ROLE OF CHANCE In vitro assays demonstrated that EV/Ecto-ESCs drove macrophages towards an M2-like phenotype. Moreover, LGMNP1 contributed to EV/Ecto-ESC-induced M2 macrophage polarization by upregulating LGMN mRNA expression levels. Clinically, serum EV-LGMNP1 was more highly expressed in recurrent EMs patients than in controls and EMs patients without recurrence. Survival analysis and our novel nomogram reconfirmed that serum EV-LGMNP1 was a novel promising and meaningful non-invasive biomarker for predicting EMs recurrence. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION In vitro experiments were only performed on samples from two patients with ovarian endometriosis, and a larger sample size is needed. ESCs isolated from the eutopic endometrium of EMs and non-EMs patients should be studied in the future. Additionally, in vitro experiments should be performed using endometrial epithelium cells and further in vivo experiments, such as using mice endometriotic models to investigate whether EV/Ecto could induce M2 macrophage polarization, should be conducted. Moreover, multicentre, large-sample data are needed to validate our predictive nomogram model. WIDER IMPLICATIONS OF THE FINDINGS Our study provides novel insights into the mechanism of M2 polarization involved in ovarian EMs progression mediated by an ‘EV-shuttled pseudogene LGMNP1’ mode. In addition, serum EV-LGMNP1 may serve as a novel non-invasive biomarker for predicting recurrence, providing a new therapeutic target for ovarian EMs. STUDY FUNDING/COMPETING INTEREST(S) This project was supported by funding from the National Natural Science Foundation of China (81971361), the Natural Science Foundation of Shanghai Science and Technology (19ZR1406900), the Shanghai ‘Rising Stars of Medical Talent’ Youth Development Program (AB83030002019004), the Clinical Research Plan of SHDC (SHDC2020CR4087), the Shanghai Municipal Health Commission (202040498), the Research and Innovation Project of the Shanghai Municipal Education Commission (2019-01-07-00-07-E00050) and the Clinical Research Plan of SHDC (SHDC2020CR1045B). There are no competing interests to declare.

LINE-1-mediated retrotransposition of protein-coding mRNAs is an active process in modern humans for both germline and somatic genomes. Prior works that surveyed human data mostly relied on detecting discordant mappings of paired-end short reads, or exon junctions contained in short reads. Moreover, there have been few genome-wide comparisons between gene retrocopies in great apes and humans. In this study, we introduced a more sensitive and accurate method to identify processed pseudogenes. Our method utilizes long-read assemblies, and more importantly, is able to provide full-length retrocopy sequences as well as flanking regions which are missed by short-read based methods. From 22 human individuals, we pinpointed 40 processed pseudogenes that are not present in the human reference genome GRCh38 and identified 17 pseudogenes that are in GRCh38 but absent from some input individuals. This represents a significantly higher discovery rate than previous reports (39 pseudogenes not in the reference genome out of 939 individuals). We also provided an overview of lineage-specific retrocopies in chimpanzee, gorilla, and orangutan genomes.

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org. National Human Genome Research Institute of the National Institutes of Health [U41HG007234]; the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health; Wellcome Trust [WT108749/Z/15/Z, WT200990/Z/16/Z]; European Molecular Biology Laboratory; Swiss National Science Foundation through the National Center of Competence in Research ‘RNA & Disease’ (to R.J.); Medical Faculty of the University of Bern (to R.J). Funding for open access charge: National Institutes of Health.

Alternative splicing (AS) and alternative polyadenylation (APA) generate diverse transcripts in mammalian genomes during development and differentiation. Epigenetic marks such as trimethylation of histone H3 lysine 36 (H3K36me3) and DNA methylation play a role in generating transcriptome diversity. Intragenic CpG islands (iCGIs) and their corresponding host genes exhibit dynamic epigenetic and gene expression patterns during development and between different tissues. We hypothesise that iCGI-associated H3K36me3, DNA methylation and transcription can influence host gene AS and/or APA. We investigate H3K36me3 and find that this histone mark is not a major regulator of AS or APA in our model system. Genomewide, we identify over 4000 host genes that harbour an iCGI in the mammalian genome, including both previously annotated and novel iCGI/host gene pairs. The transcriptional activity of these iCGIs is tissue- and developmental stage-specific and, for the first time, we demonstrate that the premature termination of host gene transcripts upstream of iCGIs is closely correlated with the level of iCGI transcription in a DNA-methylation independent manner. These studies suggest that iCGI transcription, rather than H3K36me3 or DNA methylation, interfere with host gene transcription and pre-mRNA processing genomewide and contributes to the spatiotemporal diversification of both the transcriptome and proteome.

The rapid expansion of high-quality genome assemblies, exemplified by ongoing initiatives such as the Genome-10K and i5k, demands novel automated methods to approach comparative genomics. Of these, the study of inactivating mutations in the coding region of genes, or pseudogenization, as a source of evolutionary novelty is mostly overlooked. Thus, to address such evolutionary/genomic events, a systematic, accurate and computationally automated approach is required. Here, we present PseudoChecker, the first integrated online platform for gene inactivation inference. Unlike the few existing methods, our comparative genomics-based approach displays full automation, a built-in graphical user interface and a novel index, PseudoIndex, for an empirical evaluation of the gene coding status. As a multi-platform online service, PseudoChecker simplifies access and usability, allowing a fast identification of disruptive mutations. An analysis of 30 genes previously reported to be eroded in mammals, and 30 viable genes from the same lineages, demonstrated that PseudoChecker was able to correctly infer 97% of loss events and 95% of functional genes, confirming its reliability. PseudoChecker is freely available, without login required, at http://pseudochecker.ciimar.up.pt.

Motivation Retrocopies or processed pseudogenes are gene copies resulting from mRNA retrotransposition. These gene duplicates can be fixed, somatically inserted or polymorphic in the genome. However, knowledge regarding unfixed retrocopies (retroCNVs) is still limited, and the development of computational tools for effectively identifying and genotyping them is an urgent need. Results Here, we present sideRETRO, a pipeline dedicated not only to detecting retroCNVs in whole-genome or whole-exome sequencing data but also to revealing their insertion sites, zygosity and genomic context and classifying them as somatic or polymorphic events. We show that sideRETRO can identify novel retroCNVs and genotype them, in addition to finding polymorphic retroCNVs in whole-genome and whole-exome data. Therefore, sideRETRO fills a gap in the literature and presents an efficient and straightforward algorithm to accelerate the study of bona fide retroCNVs. Availability and implementation sideRETRO is available at https://github.com/galantelab/sideRETRO Supplementary information Supplementary data are available at Bioinformatics online.

Several studies have linked mitochondrial genetic variation to phenotypic modifications; albeit the identity of the mitochondrial polymorphisms involved remains elusive. The search for these polymorphisms led to the discovery of small noncoding RNAs, which appear to be transcribed by the mitochondrial DNA (“small mitochondrial RNAs”). This contention is, however, controversial because the nuclear genome of most animals harbors mitochondrial pseudogenes (NUMTs) of identical sequence to regions of mtDNA, which could alternatively represent the source of these RNAs. To discern the likely contributions of the mitochondrial and nuclear genome to transcribing these small mitochondrial RNAs, we leverage data from six vertebrate species exhibiting markedly different levels of NUMT sequence. We explore whether abundances of small mitochondrial RNAs are associated with levels of NUMT sequence across species, or differences in tissue-specific mtDNA content within species. Evidence for the former would support the hypothesis these RNAs are primarily transcribed by NUMT sequence, whereas evidence for the latter would provide strong evidence for the counter hypothesis that these RNAs are transcribed directly by the mtDNA. No association exists between the abundance of small mitochondrial RNAs and NUMT levels across species. Moreover, a sizable proportion of transcripts map exclusively to the mtDNA sequence, even in species with highest NUMT levels. Conversely, tissue-specific abundances of small mitochondrial RNAs are strongly associated with the mtDNA content. These results support the hypothesis that small mitochondrial RNAs are primarily transcribed by the mitochondrial genome and that this capacity is conserved across Amniota and, most likely, across most metazoan lineages.

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller’s sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb–O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb–O2 affinity in (sub)Arctic Steller’s sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

Pseudogenes (Ψs), nonfunctional relatives of functional genes, form by duplication or retrotransposition, and loss of gene function by disabling mutations. Evolutionary analysis provides clues to Ψ origins and effects on gene regulation. However, few systematic studies of plant Ψs have been conducted, hampering comparative analyses. Here, we examined the origin, evolution, and expression patterns of Ψs and their relationships with noncoding sequences in seven angiosperm plants. We identified ∼250,000 Ψs, most of which are more lineage specific than protein-coding genes. The distribution of Ψs on the chromosome indicates that genome recombination may contribute to Ψ elimination. Most Ψs evolve rapidly in terms of sequence and expression levels, showing tissue- or stage-specific expression patterns. We found that a surprisingly large fraction of nontransposable element regulatory noncoding RNAs (microRNAs and long noncoding RNAs) originate from transcription of Ψ proximal upstream regions. We also found that transcription factor binding sites preferentially occur in putative Ψ proximal upstream regions compared with random intergenic regions, suggesting that Ψs have conditioned genome evolution by providing transcription factor binding sites that serve as promoters and enhancers. We therefore propose that rapid rewiring of Ψ transcriptional regulatory regions is a major mechanism driving the origin of novel regulatory modules.

The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation. Specifically, we generate primary data, create bioinformatics tools and provide analysis to support the work of expert manual gene annotators and automated gene annotation pipelines. In addition, manual and computational annotation workflows use any and all publicly available data and analysis, along with the research literature to identify and characterise gene loci to the highest standard. GENCODE gene annotations are accessible via the Ensembl and UCSC Genome Browsers, the Ensembl FTP site, Ensembl Biomart, Ensembl Perl and REST APIs as well as https://www.gencodegenes.org. We thank Tim Hubbard and Jennifer Harrow for their lead- ership in the GENCODE project from 2003-2016 as well as all groups and group members involved in the GENCODE project since its inception including the HAVANA manual annotation group formerly at Wellcome Sanger Institute now at EMBL-EBI (founder), the Guigo group at Centre for Genomic Regulation (founder), the Gerstein group at Yale (founder), the Center for Biomolecular Science & En- gineering at UCSC (founder), the Ensembl team at EMBL- EBI (joined 2007), the Kellis group at MIT (joined 2007), the Tress group at CNIO (joined 2007), the Choudhary group formerly at Wellcome Sanger Institute now at Insti- tute of Cancer Research (joined 2012), the Reymond group at University of Lausanne (2003–2017), the Antonarakis group at University of Geneva (2003–2007), the Wei group at Genome Institute of Singapore (2003–2007), the Gin- geras group at Affymetrix Ltd (2003–2007) and the Brent group at Washington University in St. Louis (2007–2012). Sí

BackgroundPolyploidy is centrally important in the evolution and domestication of plants because it leads to major genomic changes, such as altered patterns of gene expression, which are thought to underlie the emergence of new traits. Despite the common occurrence of these globally altered patterns of gene expression in polyploids, the mechanisms involved are not well understood.ResultsUsing a precisely defined framework of highly conserved syntenic genes on hexaploid wheat chromosome 3DL and its progenitor 3 L chromosome arm of diploid Aegilops tauschii, we show that 70% of these gene pairs exhibited proportionately reduced gene expression, in which expression in the hexaploid context of the 3DL genes was ∼40% of the levels observed in diploid Ae tauschii. Several genes showed elevated expression during the later stages of grain development in wheat compared with Ae tauschii. Gene sequence and methylation differences probably accounted for only a few cases of differences in gene expression. In contrast, chromosome-wide patterns of reduced chromatin accessibility of genes in the hexaploid chromosome arm compared with its diploid progenitor were correlated with both reduced gene expression and the imposition of new patterns of gene expression.ConclusionsOur pilot-scale analyses show that chromatin compaction may orchestrate reduced gene expression levels in the hexaploid chromosome arm of wheat compared to its diploid progenitor chromosome arm.

Heterotrophic plants provide evolutionarily independent, natural experiments in the genomic consequences of radically altered nutritional regimes. Here, we have sequenced and annotated the plastid genome of the endangered mycoheterotrophic orchid Hexalectris warnockii. This orchid bears a plastid genome that is ∼80% the total length of the leafy, photosynthetic Phalaenopsis, and contains just over half the number of putatively functional genes of the latter. The plastid genome of H. warnockii bears pseudogenes and has experienced losses of genes encoding proteins directly (e.g., psa/psb, rbcL) and indirectly involved in photosynthesis (atp genes), suggesting it has progressed beyond the initial stages of plastome degradation, based on previous models of plastid genome evolution. Several dispersed and tandem repeats were detected, that are potentially useful as conservation genetic markers. In addition, a 29-kb inversion and a significant contraction of the inverted repeat boundaries are observed in this plastome. The Hexalectris warnockii plastid genome adds to a growing body of data useful in refining evolutionary models in parasites, and provides a resource for conservation studies in these endangered orchids.

The enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH) is responsible for the synthesis of N-glycolylneuraminic acid (Neu5Gc), a sialic acid present on the cell surface proteins of most deuterostomes. The CMAH gene is thought to be present in most deuterostomes, but it has been inactivated in a number of lineages, including humans. The inability of humans to synthesize Neu5Gc has had several evolutionary and biomedical implications. Remarkably, Neu5Gc is a xenoantigen for humans, and consumption of Neu5Gc-containing foods, such as red meats, may promote inflammation, arthritis, and cancer. Likewise, xenotransplantation of organs producing Neu5Gc can result in inflammation and organ rejection. Therefore, knowing what animal species contain a functional CMAH gene, and are thus capable of endogenous Neu5Gc synthesis, has potentially far-reaching implications. In addition to humans, other lineages are known, or suspected, to have lost CMAH; however, to date reports of absent and pseudogenic CMAH genes are restricted to a handful of species. Here, we analyze all available genomic data for nondeuterostomes, and 322 deuterostome genomes, to ascertain the phylogenetic distribution of CMAH. Among nondeuterostomes, we found CMAH homologs in two green algae and a few prokaryotes. Within deuterostomes, putatively functional CMAH homologs are present in 184 of the studied genomes, and a total of 31 independent gene losses/pseudogenization events were inferred. Our work produces a list of animals inferred to be free from endogenous Neu5Gc based on the absence of CMAH homologs and are thus potential candidates for human consumption, xenotransplantation research, and model organisms for investigation of human diseases.

To date, little is known about the evolution of plastid genomes (plastomes) in Lauraceae. As one of the top five largest families in tropical forests, the Lauraceae contain many species that are important ecologically and economically. Lauraceous species also provide wonderful materials to study the evolutionary trajectory in response to parasitism because they contain both nonparasitic and parasitic species. This study compared the plastomes of nine Lauraceous species, including the sole hemiparasitic and herbaceous genus Cassytha (laurel dodder; here represented by Cassytha filiformis). We found differential contractions of the canonical inverted repeat (IR), resulting in two IR types present in Lauraceae. These two IR types reinforce Cryptocaryeae and Neocinnamomum—Perseeae–Laureae as two separate clades. Our data reveal several traits unique to Cas. filiformis, including loss of IRs, loss or pseudogenization of 11 ndh and rpl23 genes, richness of repeats, and accelerated rates of nucleotide substitutions in protein-coding genes. Although Cas. filiformis is low in chlorophyll content, our analysis based on dN/dS ratios suggests that both its plastid house-keeping and photosynthetic genes are under strong selective constraints. Hence, we propose that short generation time and herbaceous lifestyle rather than reduced photosynthetic ability drive the accelerated rates of nucleotide substitutions in Cas. filiformis.

Molecular species identification using suboptimal PCR primers can over-estimate species diversity due to coamplification of nuclear mitochondrial (NUMT) DNA/pseudogenes. For the agriculturally important whitefly Bemisia tabaci cryptic pest species complex, species identification depends primarily on characterization of the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene. The lack of robust PCR primers for the mtDNA COI gene can undermine correct species identification which in turn compromises management strategies. This problem is identified in the B. tabaci Africa/Middle East/Asia Minor clade which comprises the globally invasive Mediterranean (MED) and Middle East Asia Minor I (MEAM1) species, Middle East Asia Minor 2 (MEAM2), and the Indian Ocean (IO) species. Initially identified from the Indian Ocean island of Réunion, MEAM2 has since been reported from Japan, Peru, Turkey and Iraq. We identified MEAM2 individuals from a Peruvian population via Sanger sequencing of the mtDNA COI gene. In attempting to characterize the MEAM2 mitogenome, we instead characterized mitogenomes of MEAM1. We also report on the mitogenomes of MED, AUS, and IO thereby increasing genomic resources for members of this complex. Gene synteny (i.e., same gene composition and orientation) was observed with published B. tabaci cryptic species mitogenomes. Pseudogene fragments matching MEAM2 partial mtDNA COI gene exhibited low frequency single nucleotide polymorphisms that matched low copy number DNA fragments (

Functional gene transfers from the mitochondrion to the nucleus are ongoing in angiosperms and have occurred repeatedly for all 15 ribosomal protein genes, but it is not clear why some of these genes are transferred more often than others nor what the balance is between DNA- and RNA-mediated transfers. Although direct insertion of mitochondrial DNA into the nucleus occurs frequently in angiosperms, case studies of functional mitochondrial gene transfer have implicated an RNA-mediated mechanism that eliminates introns and RNA editing sites, which would otherwise impede proper expression of mitochondrial genes in the nucleus. To elucidate the mechanisms that facilitate functional gene transfers and the evolutionary dynamics of the coexisting nuclear and mitochondrial gene copies that are established during these transfers, we have analyzed rpl5 genes from 90 grasses (Poaceae) and related monocots. Multiple lines of evidence indicate that rpl5 has been functionally transferred to the nucleus at least three separate times in the grass family and that at least seven species have intact and transcribed (but not necessarily functional) copies in both the mitochondrion and nucleus. In two grasses, likely functional nuclear copies of rpl5 have been subject to recent gene conversion events via secondarily transferred mitochondrial copies in what we believe are the first described cases of mitochondrial-to-nuclear gene conversion. We show that rpl5 underwent a retroprocessing event within the mitochondrial genome early in the evolution of the grass family, which we argue predisposed the gene towards successful, DNA-mediated functional transfer by generating a “pre-edited” sequence.

UDP-glucuronosyltransferases (UGTs) are among the most important xenobiotic metabolizing enzymes that conjugate a wide range of chemicals. Previous studies showed that Felidae and Pinnipedia species have very low UGT activities toward some phenolic compounds because of the UGT1A6 pseudogene and small numbers of UGT1A isozymes. In addition to the UGT1As, UGT2Bs isozymes also conjugate various endogenous (eg, estrogens, androgens, and bile acids) and exogenous compounds (opioids, non-steroidal anti-inflammatory drugs, and environmental pollutants). However UGT2B activity and genetic background are unknown in carnivore species. Therefore, this study was performed to elucidate the species differences of UGT2Bs. Using typical substrates for UGT2Bs, UGT activity was measured in vitro. In addition, UGT2B genetic features are analyzed in silico. Results of UGT activity measurement indicate marked species differences between dogs and other carnivores (cats, Northern fur seals, Steller sea lions, Harbor seals, and Caspian seals). Dogs have very high Vmax/Km toward estradiol (17-glucuronide), estrone, lorazepam, oxazepam, and temazepam. Conversely, cats and pinniped species (especially Caspian seals and Harbor seals) have very low activities toward these substrates. The results of genetic synteny analysis indicate that Felidae and pinniped species have very small numbers of UGT2B isozymes (one or none) compared with dogs, rodents, and humans. Furthermore, Felidae species have the same nonsense mutation in UGT2B, which suggests that Felidae UGT2B31-like is also a pseudogene in addition to UGT1A6. These findings of lower activity of UGT2B suggest that Felidae and some pinniped species have very low UGT activity toward a wide range of chemicals. These results are important for Felidae and Pinnipedia species that are frequently exposed to drugs and environmental pollutants.

Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an "rDNA-like signal", representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance.

Background Long thought “relics” of evolution, not until recently have pseudogenes been of medical interest regarding regulation in cancer. Often, these regulatory roles are a direct by-product of their close sequence homology to protein-coding genes. Novel pseudogene-gene (PGG) functional associations can be identified through the integration of biomedical data, such as sequence homology, functional pathways, gene expression, pseudogene expression, and microRNA expression. However, not all of the information has been integrated, and almost all previous pseudogene studies relied on 1:1 pseudogene–parent gene relationships without leveraging other homologous genes/pseudogenes. Results We produce PGG families that expand beyond the current 1:1 paradigm. First, we construct expansive PGG databases by (i) CUDAlign graphics processing unit (GPU) accelerated local alignment of all pseudogenes to gene families (totaling 1.6 billion individual local alignments and >40,000 GPU hours) and (ii) BLAST-based assignment of pseudogenes to gene families. Second, we create an open-source web application (PseudoFuN [Pseudogene Functional Networks]) to search for integrative functional relationships of sequence homology, microRNA expression, gene expression, pseudogene expression, and gene ontology. We produce four “flavors” of CUDAlign-based databases (>462,000,000 PGG pairwise alignments and 133,770 PGG families) that can be queried and downloaded using PseudoFuN. These databases are consistent with previous 1:1 PGG annotation and also are much more powerful including millions of de novo PGG associations. For example, we find multiple known (e.g., miR-20a-PTEN-PTENP1) and novel (e.g., miR-375-SOX15-PPP4R1L) microRNA-gene-pseudogene associations in prostate cancer. PseudoFuN provides a “one stop shop” for identifying and visualizing thousands of potential regulatory relationships related to pseudogenes in The Cancer Genome Atlas cancers. Conclusions Thousands of new PGG associations can be explored in the context of microRNA-gene-pseudogene co-expression and differential expression with a simple-to-use online tool by bioinformaticians and oncologists alike.

Genome annotation, assisted by computer programs, is one of the great advances in modern biology. Nevertheless, the in silico identification of small and complex coding sequences is still challenging. We observed that amino acid sequences inferred from coding—but rarely from non-coding—DNA sequences accumulated alignments in low-stringency BLAST searches, suggesting that this alignments accumulation could be used to highlight coding regions in sequenced DNA. To investigate this possibility, we developed a computer program (AnABlast) that generates profiles of accumulated alignments in query amino acid sequences using a low-stringency BLAST strategy. To validate this approach, all six-frame translations of DNA sequences between every two annotated exons of the fission yeast genome were analysed with AnABlast. AnABlast-generated profiles identified three new copies of known genes, and four new genes supported by experimental evidence. New pseudogenes, ancestral carboxyl- and amino-terminal subtractions, complex gene rearrangements, and ancient fragments of mitDNA and of bacterial origin, were also inferred. Thus, this novel in silico approach provides a powerful tool to uncover new genes, as well as fossil-coding sequences, thus providing insight into the evolutionary history of annotated genomes.

Introduction Gnetophytes, comprising the genera Ephedra, Gnetum and Welwitschia, are an understudied, enigmatic lineage of gymnosperms with a controversial phylogenetic relationship to other seed plants. Here we examined the organization of ribosomal DNA (rDNA) across representative species. Methods We applied high-throughput sequencing approaches to isolate and reconstruct rDNA units and to determine their intragenomic homogeneity. In addition, fluorescent in situ hybridization and Southern blot hybridization techniques were used to reveal the chromosome and genomic organization of rDNA. Key results The 5S and 35S rRNA genes were separate (S-type) in Gnetum montanum, Gnetum gnemon and Welwitschia mirabilis and linked (L-type) in Ephedra altissima. There was considerable variability in 5S rDNA abundance, ranging from as few as ~4000 (W. mirabilis) to >100 000 (G. montanum) copies. A similar large variation was also observed in 5S rDNA locus numbers (two to 16 sites per diploid cell). 5S rRNA pseudogenes were interspersed between functional genes forming a single unit in E. altissima and G. montanum. Their copy number was comparable or even higher than that of functional 5S rRNA genes. In E. altissima internal transcribed spacers of 35S rDNA were long and intrinsically repetitive while in G. montanum and W. mirabilis they were short without the subrepeats. Conclusions Gnetophytes are distinct from other gymnosperms and angiosperms as they display surprisingly large variability in rDNA organization and rDNA copy and locus numbers between genera, with no relationship between copy numbers and genome sizes apparent. Concerted evolution of 5S rDNA units seems to have led to the amplification of 5S pseudogenes in both G. montanum and E. altissima. Evolutionary patterns of rDNA show both gymnosperm and angiosperm features underlining the diversity of the group.

Transposable elements, once described by Barbara McClintock as controlling genetic units, not only occupy the largest part of our genome but are also a prominent moving force of genomic plasticity and innovation. They usually replicate and reintegrate into genomes silently, sometimes causing malfunctions or misregulations, but occasionally millions of years later, a few may evolve into new functional units. Retrotransposons make their way into the genome following reverse transcription of RNA molecules and chromosomal insertion. In therian mammals, long interspersed elements 1 (LINE1s) self-propagate but also coretropose many RNAs, including mRNAs and small RNAs that usually exhibit an oligo(A) tail. The revitalization of specific LINE1 elements in the mammalian lineage about 150 Ma parallels the rise of many other nonautonomous mobilized genomic elements. We previously identified and described hundreds of tRNA-derived retropseudogenes missing characteristic oligo(A) tails consequently termed tailless retropseudogenes. Additional analyses now revealed hundreds of thousands of tailless retropseudogenes derived from nearly all types of RNAs. We extracted 2,402 perfect tailless sequences (with discernible flanking target site duplications) originating from tRNAs, spliceosomal RNAs, 5S rRNAs, 7SK RNAs, mRNAs, and others. Interestingly, all are truncated at one or more defined positions that coincide with internal single-stranded regions. 5S ribosomal and U2 spliceosomal RNAs were analyzed in the context of mammalian phylogeny to discern the origin of the therian LINE1 retropositional system that evolved in our 150-Myr-old ancestor.

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.

There is strong evidence that olfaction plays a key role in the homing of salmonids to their natal spawning grounds, particularly in the freshwater phase. However, the physiological and genetic mechanisms behind this biological phenomenon are largely unknown. It has been shown that Pacific salmon respond to dissolved free amino acids from their natal streams. This indicates that amino acids comprise part of the olfcatory cues for imprinting and homing in salmonids. As trace amine-associated receptors (TAARs), a class of olfactory receptors that are close relatives of the G protein-coupled aminergic neurotransmitter receptors, recognize amino acid metabolites, we hypothesize that TAARs play an important role in salmon homing by recognizing olfactory cues. Therefore, to better understand homing in Atlantic salmon, we set out to characterize the TAAR genes in this species. We searched the first assembly of the Atlantic salmon genome for sequences resembling TAARs previously characterized in other teleosts. We identified 27 putatively functional TAAR genes and 25 putative TAAR pseudogenes, which cluster primarily on chromosome 21 (Ssa21). Phylogenetic analysis of TAAR amino acid sequences from 15 vertebrate species revealed the TAAR gene family arose after the divergence of jawed and jawless vertebrates. The TAARs group into three classes with salmon possessing class I and class III TAARs. Within each class, evolution is characterized by species-specific gene expansions, which is in contrast to what is observed in other olfactory receptor families (e.g., OlfCs and oras).

Taste receptor genes are functionally important in animals, with a surprising exception in the bottlenose dolphin, which shows extensive losses of sweet, umami, and bitter taste receptor genes. To examine the generality of taste gene loss, we examined seven toothed whales and five baleen whales and sequenced the complete repertoire of three sweet/umami (T1Rs) and ten bitter (T2Rs) taste receptor genes. We found all amplified T1Rs and T2Rs to be pseudogenes in all 12 whales, with a shared premature stop codon in 10 of the 13 genes, which demonstrated massive losses of taste receptor genes in the common ancestor of whales. Furthermore, we analyzed three genome sequences from two toothed whales and one baleen whale and found that the sour taste marker gene Pkd2l1 is a pseudogene, whereas the candidate salty taste receptor genes are intact and putatively functional. Additionally, we examined three genes that are responsible for taste signal transduction and found the relaxation of functional constraints on taste signaling pathways along the ancestral branch leading to whales. Together, our results strongly suggest extensive losses of sweet, umami, bitter, and sour tastes in whales, and the relaxation of taste function most likely arose in the common ancestor of whales between 36 and 53 Ma. Therefore, whales represent the first animal group to lack four of five primary tastes, probably driven by the marine environment with high concentration of sodium, the feeding behavior of swallowing prey whole, and the dietary switch from plants to meat in the whale ancestor.

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.

Genome-wide association studies (GWAS) have uncovered many genetic associations for cardiovascular disease (CVD). However, data are limited regarding causal genetic variants within implicated loci. We sought to identify regulatory variants (cis- and trans-eQTLs) affecting expression levels of 93 genes selected by their proximity to SNPs with significant associations in prior GWAS for CVD traits. Expression levels were measured by qRT-PCR in leukocytes from 1846 Framingham Heart Study participants. An additive genetic model was applied to 2.5 million imputed SNPs for each gene. Approximately 45% of genes (N = 38) harbored at least one cis-eSNP after a regional multiple-test adjustment. Applying a more rigorous significance threshold (P5 × 10(-8)), we found the expression level of 10 genes was significantly associated with more than one cis-eSNP. The top cis-eSNPs for 7 of these 10 genes exhibited moderate-to-strong association with ≥ 1 CVD clinical phenotypes. Several eSNPs or proxy SNPs (r(2) = 1) were replicated by other eQTL studies. After adjusting for the lead GWAS SNPs for the 10 genes, expression variances explained by top cis-eSNPs were attenuated markedly for LPL, FADS2 and C6orf184, suggesting a shared genetic basis for the GWAS and expression trait. A significant association between cis-eSNPs, gene expression and lipid levels was discovered for LPL and C6orf184. In conclusion, strong cis-acting variants are localized within nearly half of the GWAS loci studied, with particularly strong evidence for a regulatory role of the top GWAS SNP for expression of LPL, FADS2 and C6orf184.

Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae]) and found evidence of intracellular HGT for a 2.4-kb segment of mitochondrial DNA to the rps2-rpoC2 intergenic spacer of the plastome. The transferred region contains an rpl2 pseudogene and is flanked by plastid sequence in the mitochondrial genome, including an rpoC2 pseudogene, which likely provided the mechanism for HGT back to the plastome through double-strand break repair involving homologous recombination. The plastome insertion is restricted to tribe Asclepiadeae of subfamily Asclepiadoideae, whereas the mitochondrial rpoC2 pseudogene is present throughout the subfamily, which confirms that the plastid to mitochondrial HGT event preceded the HGT to the plastome. Although the plastome insertion has been maintained in all lineages of Asclepiadoideae, it shows minimal evidence of transcription in A. syriaca and is likely nonfunctional. Furthermore, we found recent gene conversion of the mitochondrial rpoC2 pseudogene in Asclepias by the plastid gene, which reflects continued interaction of these genomes.

The highly elongated single-celled cotton fibre consists of lint and fuzz, similar to the Arabidopsis trichome. Endoreduplication is an important determinant in Arabidopsis trichome initiation and morphogenesis. Fibre development is also controlled by functional homologues of Arabidopsis trichome patterning genes, although fibre cells do not have a branched shape like trichomes. The identification and characterization of the homologues of 10 key Arabidopsis trichome branching genes in Gossypium arboreum are reported here. Nuclear ploidy of fibres was determined, and gene function in cotton callus and fibre cells was investigated. The results revealed that the nuclear DNA content was constant in fuzz, whereas a limited and reversible change occurred in lint after initiation. Gossypeum arboreum BRANCHLESS TRICHOMES (GaBLT) was not transcribed in fibres. The homologue of STICHEL (STI), which is essential for trichome branching, was a pseudogene in Gossypium. Targeted expression of GaBLT, Arabidopsis STI, and the cytokinesis-repressing GaSIAMESE in G. hirsutum fibre cells cultured in vitro resulted in branching. The findings suggest that the distinctive developmental mechanism of cotton fibres does not depend on endoreduplication. This important component may be a relic function that can be activated in fibre cells.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. In contrast with the prevalent view that Bs do not harbor genes, our recent sequence analysis revealed that Bs of rye (Secale cereale) are rich in gene-derived sequences. We compared these gene-like fragments of the rye B with their ancestral A-located counterparts and confirmed an A chromosomal origin and the pseudogenization of B-located gene-like fragments. About 15% of the pseudogene-like fragments on Bs are transcribed in a tissue-type and genotype-specific manner. In addition, B-located sequences can cause in trans down- or upregulation of A chromosome-encoded genic fragments. Phenotypes and effects associated with the presence of Bs might be explained by the activity of B-located pseudogenes. We propose a model for the evolution of B-located pseudogenes.

The POU transcription factor OCT4 is a pleiotropic regulator of gene expression in embryonic stem cells. Recent studies demonstrated that OCT4 is aberrantly expressed in multiple types of human cancer; however, the underlying molecular mechanism remains largely unknown. In this study, we report that OCT4-pg4, a pseudogene of OCT4, is abnormally activated in hepatocellular carcinoma (HCC). The expression level of OCT4-pg4 is positively correlated with that of OCT4, and both gene transcripts can be directly targeted by a tumor-suppressive micro RNA miR-145. We find that the non-coding RNA OCT4-pg4 is biologically active, as it can upregulate OCT4 protein level in HCC. Mechanistic analysis revealed that OCT4-pg4 functions as a natural micro RNA sponge to protect OCT4 transcript from being inhibited by miR-145. In addition, our study also showed that OCT4-pg4 can promote growth and tumorigenicity of HCC cells, thus exerting an oncogenic role in hepatocarcinogenesis. Furthermore, survival analysis suggests that high OCT4-pg4 level is significantly correlated with poor prognosis of HCC patients. Taken together, our finding adds a new layer of post-transcriptional regulation of OCT4 and sheds new light on the treatment of human HCC.

Long INterspersed Elements (LINE-1s, L1s) are responsible for over one million retrotransposon insertions and 8000 processed pseudogenes (PPs) in the human genome. An active L1 encodes two proteins (ORF1p and ORF2p) that bind with L1 RNA and form L1-ribonucleoprotein particles (RNPs). Although it is believed that the RNA-binding property of ORF1p is critical to recruit other mobile RNAs to the RNP, the identity of recruited RNAs is largely unknown. Here, we used crosslinking and immunoprecipitation followed by deep sequencing to identify RNA components of L1-RNPs. Our results show that in addition to retrotransposed RNAs [L1, Alu and SINE-VNTR-Alu (SVA)], L1-RNPs are enriched with cellular mRNAs, which have PPs in the human genome. Using purified L1-RNPs, we show that PP-source RNAs preferentially serve as ORF2p templates in a reverse transcriptase assay. In addition, we find that exogenous ORF2p binds endogenous ORF1p, allowing reverse transcription of the same PP-source RNAs. These data demonstrate that interaction of a cellular RNA with the L1-RNP is an inside track to PP formation.

Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene ψPPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted ψPPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected ψPPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of ψPPM1K. Furthermore, expression of NEK8 in ψPPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of ψPPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth.

The Arabidopsis (Arabidopsis thaliana) genome is the most well-annotated plant genome. However, transcriptome sequencing in Arabidopsis continues to suggest the presence of polyadenylated (polyA) transcripts originating from presumed intergenic regions. It is not clear whether these transcripts represent novel noncoding or protein-coding genes. To understand the nature of intergenic polyA transcription, we first assessed its abundance using multiple messenger RNA sequencing data sets. We found 6,545 intergenic transcribed fragments (ITFs) occupying 3.6% of Arabidopsis intergenic space. In contrast to transcribed fragments that map to protein-coding and RNA genes, most ITFs are significantly shorter, are expressed at significantly lower levels, and tend to be more data set specific. A surprisingly large number of ITFs (32.1%) may be protein coding based on evidence of translation. However, our results indicate that these “translated” ITFs tend to be close to and are likely associated with known genes. To investigate if ITFs are under selection and are functional, we assessed ITF conservation through cross-species as well as within-species comparisons. Our analysis reveals that 237 ITFs, including 49 with translation evidence, are under strong selective constraint and relatively distant from annotated features. These ITFs are likely parts of novel genes. However, the selective pressure imposed on most ITFs is similar to that of randomly selected, untranscribed intergenic sequences. Our findings indicate that despite the prevalence of ITFs, apart from the possibility of genomic contamination, many may be background or noisy transcripts derived from “junk” DNA, whose production may be inherent to the process of transcription and which, on rare occasions, may act as catalysts for the creation of novel genes.

Gene duplicates generated via retroposition were long thought to be pseudogenized and consequently decayed. However, a significant number of these genes escaped their evolutionary destiny and evolved into functional genes. Despite multiple studies, the number of functional retrogenes in human and other genomes remains unclear. We performed a comparative analysis of human, chicken, and worm genomes to identify “orphan” retrogenes, that is, retrogenes that have replaced their progenitors. We located 25 such candidates in the human genome. All of these genes were previously known, and the majority has been intensively studied. Despite this, they have never been recognized as retrogenes. Analysis revealed that the phenomenon of replacing parental genes with their retrocopies has been taking place over the entire span of animal evolution. This process was often species specific and contributed to interspecies differences. Surprisingly, these retrogenes, which should evolve in a more relaxed mode, are subject to a very strong purifying selection, which is, on average, two and a half times stronger than other human genes. Also, for retrogenes, they do not show a typical overall tendency for a testis-specific expression. Notably, seven of them are associated with human diseases. Recognizing them as “orphan” retrocopies, which have different regulatory machinery than their parents, is important for any disease studies in model organisms, especially when discoveries made in one species are transferred to humans.

The HLA region shows diversity concerning the number and content of DRB genes present per haplotype. Similar observations are made for the equivalent regions in other primate species. To elucidate the evolutionary history of the various HLA-DRB genes, a large panel of intron sequences obtained from humans, chimpanzees, rhesus macaques, and common marmosets has been subjected to phylogenetic analyses. Special attention was paid to the presence and absence of particular transposable elements and/or to their segments. The sharing of different parts of the same long interspersed nuclear element-2 (LINE2, L2) and various Alu insertions by the species studied demonstrates that one precursor gene must have been duplicated several times before the Old World monkey (OWM) and hominid (HOM) divergence. At least four ancestral DRB gene families appear to have been present before the radiation of OWM and HOM, and one of these even predates the speciation of Old and New World primates. Two of these families represent the pseudogenes DRB6/DRB2 and DRB7, which have been locked in the genomes of various primate species over long evolutionary time spans. Furthermore, all phylogenies of different intron segments show consistently that, apart from the pseudogenes, only DRB5 genes are shared by OWM and HOM, and they demonstrate the common history of certain DRB genes/lineages of humans and chimpanzees. In contrast, the evolutionary history of some other DRB loci is difficult to decipher, thus illustrating the complex history of the evolution of DRB genes due to a combination of mutations and recombination-like events. The selected approach allowed us to shed light on the ancestral DRB gene pool in primates and on the evolutionary relationship of the various HLA-DRB genes.

The olfactory receptor (OR) gene family is the largest gene family found in mammalian genomes. It is known to evolve through a birth-and-death process. Here, we characterized the sequences of 16 segregating OR pseudogenes in the samples of the wolf and the Chinese village dog (CVD) and compared them with the sequences from dogs of different breeds. Our results show that the segregating OR pseudogenes in breed dogs are under strong purifying selection, while evolving neutrally in the CVD, and show a more complicated pattern in the wolf. In the wolf, we found a trend to remove deleterious polymorphisms and accumulate nondeleterious polymorphisms. On the basis of protein structure of the ORs, we found that the distribution of different types of polymorphisms (synonymous, nonsynonymous, tolerated, and untolerated) varied greatly between the wolf and the breed dogs. In summary, our results suggest that different forms of selection have acted on the segregating OR pseudogenes in the CVD since domestication, breed dogs after breed formation, and ancestral wolf population, which has driven the evolution of these genes in different directions.

The origin and evolution of "ORFans" (suspected genes without known relatives) remain unclear. Here, we take advantage of a unique opportunity to examine the population diversity of thousands of ORFans, based on a collection of 35 complete genomes of isolates of Escherichia coli and Shigella (which is included phylogenetically within E. coli). As expected from previous studies, ORFans are shorter and AT-richer in sequence than non-ORFans. We find that ORFans often are very narrowly distributed: the most common pattern is for an ORFan to be found in only one genome. We compared within-species population diversity of ORFan genes with those of two control groups of non-ORFan genes. Patterns of population variation suggest that most ORFans are not artifacts, but encode real genes whose protein-coding capacity is conserved, reflecting selection against nonsynonymous mutations. Nevertheless, nonsynonymous nucleotide diversity is higher than for non-ORFans, whereas synonymous diversity is roughly the same. In particular, there is a several-fold excess of ORFans in the highest decile of diversity relative to controls, which might be due to weaker purifying selection, positive selection, or a subclass of ORFans that are decaying.

Several recent studies have found that selection acts on synonymous mutations at the beginning of genes to reduce mRNA secondary-structure stability, presumably to aid in translation initiation. This observation suggests that a metric of relative mRNA secondary-structure stability, Z(ΔG), could be used to test whether putative genes are likely to be functionally important. Using the Escherichia coli genome, we compared the mean Z(ΔG) of genes with known functions, genes with known orthologs, genes where function and orthology are unknown, and pseudogenes. Genes in the first two categories demonstrated similar levels of selection for reduced stability (increased Z(ΔG)), whereas for pseudogenes stability did not differ from our null expectation. Surprisingly, genes where function and orthology were unknown were also not different from the null expectation, suggesting that many of these open reading frames are not functionally important. We extended our analysis by constructing a Bayesian phylogenetic mixed model based on data from 145 prokaryotic genomes. As in E. coli, genes with no known function had consistently lower Z(ΔG), even though we expect that many of the currently unannotated genes will ultimately have their functional utility discovered. Our findings suggest that functional genes tend to evolve increased Z(ΔG), whereas nonfunctional ones do not. Therefore, Z(ΔG) may be a useful metric for identifying genes of potentially important function and could be used to target genes for further functional study.

Transfer RNAs (tRNAs) are present in all types of cells as well as in organelles. tRNAs of animal mitochondria show a low level of primary sequence conservation and exhibit 'bizarre' secondary structures, lacking complete domains of the common cloverleaf. Such sequences are hard to detect and hence frequently missed in computational analyses and mitochondrial genome annotation. Here, we introduce an automatic annotation procedure for mitochondrial tRNA genes in Metazoa based on sequence and structural information in manually curated covariance models. The method, applied to re-annotate 1876 available metazoan mitochondrial RefSeq genomes, allows to distinguish between remaining functional genes and degrading 'pseudogenes', even at early stages of divergence. The subsequent analysis of a comprehensive set of mitochondrial tRNA genes gives new insights into the evolution of structures of mitochondrial tRNA sequences as well as into the mechanisms of genome rearrangements. We find frequent losses of tRNA genes concentrated in basal Metazoa, frequent independent losses of individual parts of tRNA genes, particularly in Arthropoda, and wide-spread conserved overlaps of tRNAs in opposite reading direction. Direct evidence for several recent Tandem Duplication-Random Loss events is gained, demonstrating that this mechanism has an impact on the appearance of new mitochondrial gene orders.

IKBKG/NEMO gene mutations cause an X-linked, dominant neuroectodermal disorder named Incontinentia Pigmenti (IP). Located at Xq28, IKBKG/NEMO has a unique genomic organization, as it is part of a segmental duplication or low copy repeat (LCR1LCR2, 99 identical) containing the gene and its pseudogene copy (IKBKGP). In the opposite direction and outside LCR1, IKBKG/NEMO partially overlaps G6PD, whose mutations cause a common X-linked human enzymopathy. The two LCRs in the IKBKG/NEMO locus are able to recombine through non-allelic homologous recombination producing either a pathological recurrent exon 410 IKBKG/NEMO deletion (IKBKGdel) or benign small copy number variations. We here report that the local high frequency of micro/macro-homologies, tandem repeats and repeat/repetitive sequences make the IKBKG/NEMO locus susceptible to novel pathological IP alterations. Indeed, we describe the first two independent instances of inter-locus gene conversion, occurring between the two LCRs, that copies the IKBKGP pseudogene variants into the functional IKBKG/NEMO, causing the de novo occurrence of p.Glu390ArgfsX61 and the IKBKGdel mutations, respectively. Subsequently, by investigating a group of 20 molecularly unsolved IP subjects using a high-density quantitative polymerase chain reaction assay, we have identified seven unique de novo deletions varying from 4.8 to approximate to 115 kb in length. Each deletion removes partially or completely both IKBKG/NEMO and the overlapping G6PD, thereby uncovering the first deletions disrupting the G6PD gene which were found in patients with IP. Interestingly, the 4.8 kb deletion removes the conserved bidirectional promoterB, shared by the two overlapping IKBKG/NEMO and G6PD genes, leaving intact the alternative IKBKG/NEMO unidirectional promoterA. This promoter, although active in the keratinocytes of the basal dermal layer, is down-regulated during late differentiation. Genomic analysis at the breakpoint sites indicated that other mutational forces, such as non-homologous end joining, Alu-Alu-mediated recombination and replication-based events, might enhance the vulnerability of the IP locus to produce de novo pathological IP alleles.

Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ∼100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.

Almost 50 years following the discovery of the prokaryotic operon, the functional relevance of gene order within operons remains unclear. In this work, we take advantage of the eroded genome of Mycobacterium leprae to add evidence supporting the notion that functionally less important genes have a tendency to be located at the end of its operons. M. leprae’s genome includes 1133 pseudogenes and 1614 protein-coding genes and can be compared with the close genome of M. tuberculosis. Assuming M. leprae’s pseudogenes to represent dispensable genes, we have studied the position of these pseudogenes in the operons of M. leprae and of their orthologs in M. tuberculosis .W e observed that both tend to be located in the 3 0 (downstream) half of the operon (P-values of 0.03 and 0.18, respectively). Analysis of pseudogenes in all available prokaryotic genomes confirms this trend (P-value of 7.1 � 10 � 7 ). In a complementary analysis, we found a significant tendency for essential genes to be located at the 5 0 (upstream) half of the operon (P-value of 0.006). Our work provides an indication that, in prokarya, functionally less important genes have a tendency to be located at the end of operons, while more relevant genes tend to be located toward operon starts.

Although it belongs to the order Carnivora, the giant panda is a vegetarian with 99% of its diet being bamboo. The draft genome sequence of the giant panda shows that its umami taste receptor gene Tas1r1 is a pseudogene, prompting the proposal that the loss of the umami perception explains why the giant panda is herbivorous. To test this hypothesis, we sequenced all six exons of Tas1r1 in another individual of the giant panda and five other carnivores. We found that the open reading frame (ORF) of Tas1r1 is intact in all these carnivores except the giant panda. The rate ratio (ω) of nonsynonymous to synonymous substitutions in Tas1r1 is significantly higher for the giant panda lineage than for other carnivore lineages. Based on the ω change and the observed number of ORF-disrupting substitutions, we estimated that the functional constraint on the giant panda Tas1r1 was relaxed ∼ 4.2 Ma, with its 95% confidence interval between 1.3 and 10 Ma. Our estimate matches the approximate date of the giant panda's dietary switch inferred from fossil records. It is probable that the giant panda's decreased reliance on meat resulted in the dispensability of the umami taste, leading to Tas1r1 pseudogenization, which in turn reinforced its herbivorous life style because of the diminished attraction of returning to meat eating in the absence of Tas1r1. Nonetheless, additional factors are likely involved because herbivores such as cow and horse still retain an intact Tas1r1.

Motivation: Recent years have seen the development of a wide range of biomedical ontologies. Notable among these is Sequence Ontology (SO) which offers a rich hierarchy of terms and relationships that can be used to annotate genomic data. Well-designed formal ontologies allow data to be reasoned upon in a consistent and logically sound way and can lead to the discovery of new relationships. The Semantic Web Rules Language (SWRL) augments the capabilities of a reasoner by allowing the creation of conditional rules. To date, however, formal reasoning, especially the use of SWRL rules, has not been widely used in biomedicine. Results: We have built a knowledge base of human pseudogenes, extending the existing SO framework to incorporate additional attributes. In particular, we have defined the relationships between pseudogenes and segmental duplications. We then created a series of logical rules using SWRL to answer research questions and to annotate our pseudogenes appropriately. Finally, we were left with a knowledge base which could be queried to discover information about human pseudogene evolution. Availability: The fully populated knowledge base described in this document is available for download from http://ontology.pseudogene.org. A SPARQL endpoint from which to query the dataset is also available at this location. Contact: matthew.holford@yale.edu; mark.gerstein@yale.edu