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SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17β-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.

The cricetine rodents Peromyscus leucopus and P. maniculatus are key reservoirs for several zoonotic diseases in North America. We determined the complete circular mitochondrial genome sequences of representatives of 3 different stock colonies of P. leucopus, one stock colony of P. maniculatus and two wild populations of P. leucopus. The genomes were syntenic with that of the murids Mus musculus and Rattus norvegicus. Phylogenetic analysis confirmed that these two Peromyscus species are sister taxa in a clade with P. polionotus and also uncovered a distinction between P. leucopus populations in the eastern and the central United States. In one P. leucopus lineage four extended regions of mitochondrial pseudogenes were identified in the nuclear genome. RNA-seq analysis revealed transcription of the entire genome and differences from controls in the expression profiles of mitochondrial genes in the blood, but not in liver or brain, of animals infected with the zoonotic pathogen Borrelia hermsii. PCR and sequencing of the D-loop of the mitochondrion identified 32 different haplotypes among 118 wild P. leucopus at a Connecticut field site. These findings help to further establish P. leucopus as a model organism for studies of emerging infectious diseases, ecology, and in other disciplines.

BackgroundGenome duplication has played a pivotal role in the evolution of many eukaryotic lineages, including the vertebrates. A relatively recent vertebrate genome duplication is that in Xenopus laevis, which resulted from the hybridization of two closely related species about 17 million years ago. However, little is known about the consequences of this duplication at the level of the genome, the epigenome, and gene expression.ResultsThe X. laevis genome consists of two subgenomes, referred to as L (long chromosomes) and S (short chromosomes), that originated from distinct diploid progenitors. Of the parental subgenomes, S chromosomes have degraded faster than L chromosomes from the point of genome duplication until the present day. Deletions appear to have the largest effect on pseudogene formation and loss of regulatory regions. Deleted regions are enriched for long DNA repeats and the flanking regions have high alignment scores, suggesting that non-allelic homologous recombination has played a significant role in the loss of DNA. To assess innovations in the X. laevis subgenomes we examined p300-bound enhancer peaks that are unique to one subgenome and absent from X. tropicalis. A large majority of new enhancers comprise transposable elements. Finally, to dissect early and late events following interspecific hybridization, we examined the epigenome and the enhancer landscape in X. tropicalis × X. laevis hybrid embryos. Strikingly, young X. tropicalis DNA transposons are derepressed and recruit p300 in hybrid embryos.ConclusionsThe results show that erosion of X. laevis genes and functional regulatory elements is associated with repeats and non-allelic homologous recombination and furthermore that young repeats have also contributed to the p300-bound regulatory landscape following hybridization and whole-genome duplication.

Numerous animal lineages have expanded and diversified the opsin-based photoreceptors in their eyes underlying color vision behavior. However, the selective pressures giving rise to new photoreceptors and their spectral tuning remain mostly obscure. Previously, we identified a violet receptor (UV2) that is the result of a UV opsin gene duplication specific to Heliconius butterflies. At the same time the violet receptor evolved, Heliconius evolved UV-yellow coloration on their wings, due to the pigment 3-hydroxykynurenine (3-OHK) and the nanostructure architecture of the scale cells. In order to better understand the selective pressures giving rise to the violet receptor, we characterized opsin expression patterns using immunostaining (14 species) and RNA-Seq (18 species), and reconstructed evolutionary histories of visual traits in five major lineages within Heliconius and one species from the genus Eueides. Opsin expression patterns are hyperdiverse within Heliconius. We identified six unique retinal mosaics and three distinct forms of sexual dimorphism based on ommatidial types within the genus Heliconius. Additionally, phylogenetic analysis revealed independent losses of opsin expression, pseudogenization events, and relaxation of selection on UVRh2 in one lineage. Despite this diversity, the newly evolved violet receptor is retained across most species and sexes surveyed. Discriminability modeling of behaviorally preferred 3-OHK yellow wing coloration suggests that the violet receptor may facilitate Heliconius color vision in the context of conspecific recognition. Our observations give insights into the selective pressures underlying the origins of new visual receptors.

Extracellular factors belonging to the TGF-β family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-β family genes in response to allotetraploidization.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

BackgroundDNA cytosine methylation is an important epigenetic modification that has significant effects on a variety of biological processes in animals. Avian species hold a crucial position in evolutionary history. In this study, we used whole-genome bisulfite sequencing (MethylC-seq) to generate single base methylation profiles of lungs in two genetically distinct and highly inbred chicken lines (Fayoumi and Leghorn) that differ in genetic resistance to multiple pathogens, and we explored the potential regulatory role of DNA methylation associated with immune response differences between the two chicken lines.MethodsThe MethylC-seq was used to generate single base DNA methylation profiles of Fayoumi and Leghorn birds. In addition, transcriptome profiling using RNA-seq from the same chickens and tissues were obtained to interrogate how DNA methylation regulates gene transcription on a genome-wide scale.ResultsThe general DNA methylation pattern across different regions of genes was conserved compared to other species except for hyper-methylation of repeat elements, which was not observed in chicken. The methylation level of miRNA and pseudogene promoters was high, which indicates that silencing of these genes may be partially due to promoter hyper-methylation. Interestingly, the promoter regions of more recently evolved genes tended to be more highly methylated, whereas the gene body regions of evolutionarily conserved genes were more highly methylated than those of more recently evolved genes. Immune-related GO (Gene Ontology) terms were significantly enriched from genes within the differentially methylated regions (DMR) between Fayoumi and Leghorn, which implicates DNA methylation as one of the regulatory mechanisms modulating immune response differences between these lines.ConclusionsThis study establishes a single-base resolution DNA methylation profile of chicken lung and suggests a regulatory role of DNA methylation in controlling gene expression and maintaining genome transcription stability. Furthermore, profiling the DNA methylomes of two genetic lines that differ in disease resistance provides a unique opportunity to investigate the potential role of DNA methylation in host disease resistance. Our study provides a foundation for future studies on epigenetic modulation of host immune response to pathogens in chickens.

Rod monochromacy is a rare condition in vertebrates characterized by the absence of cone photoreceptor cells. The resulting phenotype is colourblindness and low acuity vision in dim-light and blindness in bright-light conditions. Early reports of xenarthrans (armadillos, sloths and anteaters) suggest that they are rod monochromats, but this has not been tested with genomic data. We searched the genomes of Dasypus novemcinctus (nine-banded armadillo), Choloepus hoffmanni (Hoffmann's two-toed sloth) and Mylodon darwinii (extinct ground sloth) for retinal photoreceptor genes and examined them for inactivating mutations. We performed PCR and Sanger sequencing on cone phototransduction genes of 10 additional xenarthrans to test for shared inactivating mutations and estimated the timing of inactivation for photoreceptor pseudogenes. We concluded that a stem xenarthran became an long-wavelength sensitive-cone monochromat following a missense mutation at a critical residue in SWS1, and a stem cingulate (armadillos, glyptodonts and pampatheres) and stem pilosan (sloths and anteaters) independently acquired rod monochromacy early in their evolutionary history following the inactivation of LWS and PDE6C, respectively. We hypothesize that rod monochromacy in armadillos and pilosans evolved as an adaptation to a subterranean habitat in the early history of Xenarthra. The presence of rod monochromacy has major implications for understanding xenarthran behavioural ecology and evolution.

Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs (e.g., tRNAs) and more recently discovered ncRNAs (e.g., snoRNAs and miRNAs) in birds. Furthermore, we describe numerous "losses" of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes.

Interferon lambda 4 gene (IFNL4) encodes IFN-λ4, a new member of the IFN-λ family with antiviral activity. In humans IFNL4 open reading frame is truncated by a polymorphic frame-shift insertion that eliminates IFN-λ4 and turns IFNL4 into a polymorphic pseudogene. Functional IFN-λ4 has antiviral activity but the elimination of IFN-λ4 through pseudogenization is strongly associated with improved clearance of hepatitis C virus (HCV) infection. We show that functional IFN-λ4 is conserved and evolutionarily constrained in mammals and thus functionally relevant. However, the pseudogene has reached moderately high frequency in Africa, America, and Europe, and near fixation in East Asia. In fact, the pseudogenizing variant is among the 0.8% most differentiated SNPs between Africa and East Asia genome-wide. Its raise in frequency is associated with additional evidence of positive selection, which is strongest in East Asia, where this variant falls in the 0.5% tail of SNPs with strongest signatures of recent positive selection genome-wide. Using a new Approximate Bayesian Computation (ABC) approach we infer that the pseudogenizing allele appeared just before the out-of-Africa migration and was immediately targeted by moderate positive selection; selection subsequently strengthened in European and Asian populations resulting in the high frequency observed today. This provides evidence for a changing adaptive process that, by favoring IFN-λ4 inactivation, has shaped present-day phenotypic diversity and susceptibility to disease.

Mapping reads to a reference sequence is a common step when analyzing allele effects in high-throughput sequencing data. The choice of reference is critical because its effect on quantitative sequence analysis is non-negligible. Recent studies suggest aligning to a single standard reference sequence, as is common practice, can lead to an underlying bias depending on the genetic distances of the target sequences from the reference. To avoid this bias, researchers have resorted to using modified reference sequences. Even with this improvement, various limitations and problems remain unsolved, which include reduced mapping ratios, shifts in read mappings and the selection of which variants to include to remove biases. To address these issues, we propose a novel and generic multi-alignment pipeline. Our pipeline integrates the genomic variations from known or suspected founders into separate reference sequences and performs alignments to each one. By mapping reads to multiple reference sequences and merging them afterward, we are able to rescue more reads and diminish the bias caused by using a single common reference. Moreover, the genomic origin of each read is determined and annotated during the merging process, providing a better source of information to assess differential expression than simple allele queries at known variant positions. Using RNA-seq of a diallel cross, we compare our pipeline with the single-reference pipeline and demonstrate our advantages of more aligned reads and a higher percentage of reads with assigned origins. Database URL: http://csbio.unc.edu/CCstatus/index.py?run=Pseudo.

Spectral entropy and GC content analyses reveal comprehensive structural features of DNA sequences. To illustrate the significance of these features, we analyze the β-esterase gene cluster, including the Est-6 gene and the ψEst-6 putative pseudogene, in seven species of the Drosophila melanogaster subgroup. The spectral entropies show distinctly lower structural ordering for ψEst-6 than for Est-6 in all species studied. However, entropy accumulation is not a completely random process for either gene and it shows to be nucleotide dependent. Furthermore, GC content in synonymous positions is uniformly higher in Est-6 than in ψEst-6, in agreement with the reduced GC content generally observed in pseudogenes and nonfunctional sequences. The observed differences in entropy and GC content reflect an evolutionary shift associated with the process of pseudogenization and subsequent functional divergence of ψEst-6 and Est-6 after the duplication event. The data obtained show the relevance and significance of entropy and GC content analyses for pseudogene identification and for the comparative study of gene-pseudogene evolution.

BackgroundWe describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.ResultsOur phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.ConclusionsOur comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

BackgroundGene knockouts in a model organism such as mouse provide a valuable resource for the study of basic biology and human disease. Determining which gene has been inactivated by an untargeted gene trapping event poses a challenging annotation problem because gene trap sequence tags, which represent sequence near the vector insertion site of a trapped gene, are typically short and often contain unresolved residues. To understand better the localization of these sequences on the mouse genome, we compared stand-alone versions of the alignment programs BLAT, SSAHA, and MegaBLAST. A set of 3,369 sequence tags was aligned to build 34 of the mouse genome using default parameters for each algorithm. Known genome coordinates for the cognate set of full-length genes (1,659 sequences) were used to evaluate localization results.ResultsIn general, all three programs performed well in terms of localizing sequences to a general region of the genome, with only relatively subtle errors identified for a small proportion of the sequence tags. However, large differences in performance were noted with regard to correctly identifying exon boundaries. BLAT correctly identified the vast majority of exon boundaries, while SSAHA and MegaBLAST missed the majority of exon boundaries. SSAHA consistently reported the fewest false positives and is the fastest algorithm. MegaBLAST was comparable to BLAT in speed, but was the most susceptible to localizing sequence tags incorrectly to pseudogenes.ConclusionThe differences in performance for sequence tags and full-length reference sequences were surprisingly small. Characteristic variations in localization results for each program were noted that affect the localization of sequence at exon boundaries, in particular.

We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.

Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.

Efforts to understand the genetic basis of evolutionary change have concentrated on proteins and their encoding DNA sequences. These studies have brought to light patterns and processes at the nucleotide level, yet the complex functional relationships between genetic variants and phenotypes remain poorly known. The realization that even a complete description of proteins and the effects of their activity will not suffice to understand the conditions under which they are time- and tissue-specifically expressed or repressed during development has refocused attention on cis-regulatory regions. In particular, promoter sequences are thought to hold the key for understanding the evolution of phenotypic differences between species. This is because of their complex organization into independent modules such that, unlike coding sequences in which mutations affect protein function every time the protein is expressed, mutations in cis-regulatory sequences may have minor or no pleiotropic effects. Complex information-encoding makes cis-regulatory regions poorly amenable to comparative methods designed for coding sequences. Some general conclusions are emerging as to how genetic variation is distributed across regulatory networks and the processes modulating the structure of this variation. We bring into this emerging scenario several recent findings pointing to different ways in which spliceosomal introns, pseudogenes and patterns of point mutation can be active players for the evolution of novel transcriptional profiles.

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.

We have amplified and sequenced the gene encoding Esterase-P (Est-P) in 10 strains of Drosophila melanogaster. Three premature termination codons occur in the coding region of the gene in two strains. This observation, together with other indirect evidence, leads us to propose that Est-P may be a pseudogene in D. melanogaster. Est-P would be a "cryptic" pseudogene, in the sense that it retains intact the coding sequence (without stop codons and other alterations usually observed in pseudogenes) in most D. melanogaster strains. We conjecture that the beta-esterase cluster may consist in other Drosophila species of functional and nonfunctional genes. We also conjecture that the rarity of detected pseudogenes in Drosophila may be due to the difficulty of discovering them, because most of them are cryptic.

DNA methylation is mediated by a conserved family of DNA methyltransferases (Dnmts). The human genome encodes three active Dnmts (Dnmt1, Dnmt3a and Dnmt3b), the tRNA methyltransferase Dnmt2, and the regulatory protein Dnmt3L. Despite their high degree of conservation among different species, genes encoding Dnmts have been duplicated and/or lost in multiple lineages throughout evolution, indicating that the DNA methylation machinery has some potential to undergo evolutionary change. However, little is known about the extent to which this machinery, or the methylome, varies among vertebrates. Here, we study the molecular evolution of Dnmt1, the enzyme responsible for maintenance of DNA methylation patterns after replication, in 79 vertebrate species. Our analyses show that all studied species exhibit a single copy of the DNMT1 gene, with the exception of tilapia and marsupials (tammar wallaby, koala, Tasmanian devil and opossum), each of which displays two apparently functional DNMT1 copies. Our phylogenetic analyses indicate that DNMT1 duplicated before the radiation of major marsupial groups (i.e., at least ~75 million years ago), thus giving rise to two DNMT1 copies in marsupials (copy 1 and copy 2). In the opossum lineage, copy 2 was lost, and copy 1 recently duplicated again, generating three DNMT1 copies: two putatively functional genes (copy 1a and 1b) and one pseudogene (copy 1ψ). Both marsupial copies (DNMT1 copies 1 and 2) are under purifying selection, and copy 2 exhibits elevated rates of evolution and signatures of positive selection, suggesting a scenario of neofunctionalization. This gene duplication might have resulted in modifications in marsupial methylomes and their dynamics. [ABSTRACT FROM AUTHOR]

In this paper, we compare ndh genes in the plastid genome of many Cymbidium species and three closely related taxa in Orchidaceae looking for evidence of ndh gene degradation. Among the 11 ndh genes, there were frequently large deletions in directly repeated or AT-rich regions. Variation in these degraded ndh genes occurs between individual plants, apparently at population levels in these Cymbidium species. It is likely that ndh gene transfers from the plastome to mitochondrial genome (chondriome) occurred independently in Orchidaceae and that ndh genes in the chondriome were also relatively recently transferred between distantly related species in Orchidaceae. Four variants of the ycf1-rpl32 region, which normally includes the ndhF genes in the plastome, were identified, and some Cymbidium species contained at least two copies of that region in their organellar genomes. The four ycf1-rpl32 variants seem to have a clear pattern of close relationships. Patterns of ndh degradation between closely related taxa and translocation of ndh genes to the chondriome in Cymbidium suggest that there have been multiple bidirectional intracellular gene transfers between two organellar genomes, which have produced different levels of ndh gene degradation among even closely related species. [ABSTRACT FROM AUTHOR]

Keratins represent a large protein family with essential structural and functional roles in epithelial cells of skin, hair follicles, and other organs. During evolution the genes encoding keratins have undergone multiple rounds of duplication and humans have two clusters with a total of 55 functional keratin genes in their genomes. Due to the high similarity between different keratin paralogs and species-specific differences in gene content, the currently available keratin gene annotation in species with draft genome assemblies such as dog and horse is still imperfect. We compared the National Center for Biotechnology Information (NCBI) (dog annotation release 103, horse annotation release 101) and Ensembl (release 87) gene predictions for the canine and equine keratin gene clusters to RNA-seq data that were generated from adult skin of five dogs and two horses and from adult hair follicle tissue of one dog. Taking into consideration the knowledge on the conserved exon/intron structure of keratin genes, we annotated 61 putatively functional keratin genes in both the dog and horse, respectively. Subsequently, curators in the RefSeq group at NCBI reviewed their annotation of keratin genes in the dog and horse genomes (Annotation Release 104 and Annotation Release 102, respectively) and updated annotation and gene nomenclature of several keratin genes. The updates are now available in the NCBI Gene database (). [ABSTRACT FROM AUTHOR]

By searching a clinical database of over 60,000 individuals referred for array-based CNV analyses and online resources, we identified four males from three families with intellectual disability, developmental delay, hypotonia, joint hypermobility and relative macrocephaly who carried small, overlapping deletions of Xp11.22. The maximum region of overlap between their deletions spanned ~430 kb and included two pseudogenes, CENPVL1 and CENPVL2, whose functions are not known, and two protein coding genes—the G1 to S phase transition 2 gene (GSPT2) and the MAGE family member D1 gene (MAGED1). Deletions of this ~430 kb region have not been previously implicated in human disease. Duplications of GSPT2 have been documented in individuals with intellectual disability, but the phenotypic consequences of a loss of GSPT2 function have not been elucidated in humans or mouse models. Changes in MAGED1 have not been associated with intellectual disability in humans, but loss of MAGED1 function is associated with neurocognitive and neurobehavioral phenotypes in mice. In all cases, the Xp11.22 deletion was inherited from an unaffected mother. Studies performed on DNA from one of these mothers did not show evidence of skewed X-inactivation. These results suggest that deletions of an ~430 kb region on chromosome Xp11.22 that encompass CENPVL1, CENPVL2, GSPT2 and MAGED1 cause a distinct X-linked syndrome characterized by intellectual disability, developmental delay, hypotonia, joint hypermobility and relative macrocephaly. Loss of GSPT2 and/or MAGED1 function may contribute to the intellectual disability and developmental delay seen in males with these deletions. [ABSTRACT FROM AUTHOR]

A portion of the mitochondrial cytochrome c oxidase I gene was sequenced using both genomic DNA and complement DNA from three planktonic copepod Neocalanus species (N. cristatus, N. plumchrus, and N. flemingeri). Small but critical sequence differences in CO1 were observed between gDNA and cDNA from N. plumchrus. Furthermore, careful observation revealed the presence of recombination between sequences in gDNA from N. plumchrus. Moreover, a chimera of the N. cristatus and N. plumchrus sequences was obtained from N. plumchrus gDNA. The observed phenomena can be best explained by the preferential amplification of the nuclear mitochondrial pseudogenes from gDNA of N. plumchrus. Two conclusions can be drawn from the observations. First, nuclear mitochondrial pseudogenes are pervasive in N. plumchrus. Second, a mating between a female N. cristatus and a male N. plumchrus produced viable offspring, which further backcrossed to a N. plumchrus individual. These observations not only demonstrate intriguing mating behavior in these species, but also emphasize the importance of careful interpretation of species marker sequences amplified from gDNA. [ABSTRACT FROM AUTHOR]

Since the discovery of the high abundance of Alu elements in the human genome, the interest for the functional significance of these retrotransposons has been increasing. Primate Alu and rodent Alu-like elements are retrotransposed by a mechanism driven by the LINE1 (L1) encoded proteins, the same machinery that generates the L1 repeats, the processed pseudogenes (PPs), and other retroelements. Apart from free Alu RNAs, Alus are also transcribed and retrotranscribed as part of cellular gene transcripts, generally embedded inside 3’ untranslated regions (UTRs). Despite different proposed hypotheses, the functional implication of the presence of Alus inside 3’UTRs remains elusive. In this study we hypothesized that Alu elements in 3’UTRs could be involved in the genesis of PPs. By analyzing human genome data we discovered that the existence of 3’UTR-embedded Alu elements is overrepresented in genes source of PPs. In contrast, the presence of other retrotransposable elements in 3’UTRs does not show this PP linked overrepresentation. This research was extended to mouse and rat genomes and the results accordingly reveal overrepresentation of 3’UTR-embedded B1 (Alu-like) elements in PP parent genes. Interestingly, we also demonstrated that the overrepresentation of 3’UTR-embedded Alus is particularly significant in PP parent genes with low germline gene expression level. Finally, we provide data that support the hypothesis that the L1 machinery is also the system that herpesviruses, and possibly other large DNA viruses, use to capture host genes expressed in germline or somatic cells. Altogether our results suggest a novel role for Alu or Alu-like elements inside 3’UTRs as facilitators of the genesis of PPs, particularly in lowly expressed genes. Moreover, we propose that this L1-driven mechanism, aided by the presence of 3’UTR-embedded Alus, may also be exploited by DNA viruses to incorporate host genes to their viral genomes. [ABSTRACT FROM AUTHOR]

Phosphatase and tensin homolog (PTEN) is a tumor-suppressor gene. PTEN pseudogene (PTENp) acts as an endogenous RNA, which regulates its parental gene by competitively binding to the 3’ UTR of PTEN gene in the human. Despite the importance of this pseudogene, little is known about the molecular evolution of PTENp in mammals. In this study, we identified 37 pseudogenes from 65 mammalian genomes. Among them, 32 were from rodents or primates. Phylogenetic analyse showed a complex evolutionary history of this gene family. Some PTENps were shared both in primates and rodents. However, some PTENps were shown to be species-specific, such as the tasmanian devil PTENp1, nine banded armadillo PTENp1 and gibbon PTENp1. Most interestingly, the naked mole rat (NMR), an anticancer model organism, possessed 17 copies of PTENps, which were classified into four clades based on the phylogenetic analyses. Furthermore, we found that all the 3’UTR of PTEN and PTENps shared common microRNA (MicroRNA) binding sites in NMR, based on our prediction of specific MicroRNA binding sites. Our findings suggested that multiple gene duplications have occurred in the formation of PTEN/PTENp gene family during the evolution of mammals. Some PTENps were relatively ancient and were shared by primates and rodents; others were newly originated through species- specific gene duplications. PTENps in NMR may function as competitive endogenous RNAs (ceRNAs) to regulate their counterpart genes by competing for common MicroRNAs, which may be one of the interpretations for the cancer resistance in NMR. [ABSTRACT FROM AUTHOR]

We have developed a new bioinformatics framework for the analysis of rearranged bovine heavy chain immunoglobulin (Ig) variable regions by combining and refining widely used alignment algorithms. This bioinformatics framework allowed us to investigate alignments of heavy chain framework regions (FRHs) and the separate alignments of FRHs and heavy chain complementarity determining regions (CDRHs) to determine their germline origin in the four cattle breeds Aubrac, German Black Pied, German Simmental, and Holstein Friesian. Now it is also possible to specifically analyze Ig heavy chains possessing exceptionally long CDR3Hs. In order to gain more insight into breed specific differences in Ig combinatorial diversity, somatic hypermutations and putative gene conversions of IgG, we compared the dominantly transcribed variable (IGHV), diversity (IGHD), and joining (IGHJ) segments and their recombination in the four cattle breeds. The analysis revealed the use of 15 different IGHV segments, 21 IGHD segments, and two IGHJ segments with significant different transcription levels within the breeds. Furthermore, there are preferred rearrangements within the three groups of CDR3H lengths. In the sequences of group 2 (CDR3H lengths (L) of 11–47 amino acid residues (aa)) a higher number of recombination was observed than in sequences of group 1 (L≤10 aa) and 3 (L≥48 aa). The combinatorial diversity of germline IGHV, IGHD, and IGHJ-segments revealed 162 rearrangements that were significantly different. The few preferably rearranged gene segments within group 3 CDR3H regions may indicate specialized antibodies because this length is unique in cattle. The most important finding of this study, which was enabled by using the bioinformatics framework, is the discovery of strong evidence for gene conversion as a rare event using pseudogenes fulfilling all definitions for this particular diversification mechanism. [ABSTRACT FROM AUTHOR]