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Transcriptome analyses based on high-throughput RNA sequencing (RNA-Seq) provide powerful and quantitative characterization of cell types and in-depth understanding of biological systems in health and disease. In this study, we present a comprehensive transcriptome profile of human primary monocytes, a crucial component of the innate immune system. We performed deep RNA-Seq of monocytes from six healthy subjects and integrated our data with 10 other publicly available RNA-Seq datasets of human monocytes. A total of 1.9 billion reads were generated, which allowed us to capture most of the genes transcribed in human monocytes, including 11,994 protein-coding genes, 5558 noncoding genes (including long noncoding RNAs, precursor miRNAs, and others), 2819 pseudogenes, and 7034 putative novel transcripts. In addition, we profiled the expression pattern of 1155 transcription factors (TFs) in human monocytes, which are the main molecules in controlling the gene transcription. An interaction network was constructed among the top expressed TFs and their targeted genes, which revealed the potential key regulatory genes in biological function of human monocytes. The gene catalog of human primary monocytes provided in this study offers significant promise and future potential clinical applications in the fields of precision medicine, systems diagnostics, immunogenomics, and the development of innovative biomarkers and therapeutic monitoring strategies.

Little attention has been paid to the histone H2A/K pseudogene. Results from our laboratory showed that 7 of 10 kidney cancer patients carried a mutant H2A/K pseudogene; therefore, we were interested in determining the relationship between mutant H2A/K and cell proliferation. We used shotgun and label-free proteomics methods to study whether mutant H2A/K lncRNAs affected cell proliferation. Quantitative proteomic analysis indicated that the expression of mutant H2A/K lncRNAs resulted in the upregulation of many oncogenes, which promoted cell proliferation. Further interaction analyses revealed that a proliferating cell nuclear antigen (PCNA)-protein interaction network, with PCNA in the center, contributes to cell proliferation in cells expressing the mutant H2A/K lncRNAs. Western blotting confirmed the critical upregulation of PCNA by mutant H2A/K lncRNA expression. Finally, the promotion of cell proliferation by mutant H2A/K lncRNAs (C290T, C228A and A45G) was confirmed using cell proliferation assays. Although we did not determine the exact mechanism by which the oncogenes were upregulated by the mutant H2A/K lncRNAs, we confirmed that the mutant H2A/K lncRNAs promoted cell proliferation by upregulating PCNA and other oncogenes. The hypothesis that cell proliferation is promoted by the mutant H2A/K lncRNAs was supported by the protein expression and cell proliferation assay results. Therefore, mutant H2A/K lncRNAs may be a new factor in renal carcinogenesis.

Among the various areas of recent investigation in the field of human MHC class I antigens, the following have been selected for discussion in this review: (1) classical HLA class I genes: are they ubiquitously expressed?, what are the special features of their polymorphism?, are HLA-C molecules functional?, (2) non-classical HLA class I gene products: how restricted is their tissue distribution?, do they exhibit a little polymorphism?, what is their function, if any? (3) non-HLA genes recently detected in the HLA class I chromosomal region: are some of them involved in immunological function and development?, (4) other novel coding sequences present, or possibly present, in the region: the hemochromatosis gene, grc region and associated tumor suppressor genes, housekeeping genes, human equivalent of the murine H-2M region and Ped gene; (5) transcriptional regulation: are there cis-regulatory elements, including locus control region(s). located elsewhere than in the promoters? are CpG methylation, gene imprinting, chromatin structure, DNA rearrangement also implicated? what are the transcription factors involved and how do they interact with each other? is there HLA class I locus-, allele-, or isoform-specific regulation? is class I gene expression dysregulated in human tumors? The answers to these questions are crucial for the development of the future directions for research.

The nucleotide sequence of U11 small nuclear RNA, a minor U RNA from HeLa cells, was determined. Computer analysis of the sequence (135 residues) predicts two strong hairpin loops which are separated by seventeen nucleotides containing an Sm binding site (AAUUUUUUGG). A synthetic gene was constructed in which the coding region of U11 RNA is under the control of a T7 promoter. This vector can be used to produce U11 RNA in vitro. Southern hybridization and PCR analysis of HeLa genomic DNA suggest that U11 RNA is encoded by a single copy gene, and that at least three genomic regions could be U11 RNA pseudogenes. A HeLa genomic copy of a U11 gene was isolated by inverted PCR. This gene contains the U11 RNA coding sequence and several sequence elements unique for the U RNA genes. These include a Distal Sequence Element (DSE, ATTTGCATA) present between positions -215 and -223 relative to the start of transcription; a Proximal Sequence Element (PSE, TTCACCTTTACCAAAAATG) located between positions -43 and -63; and a 3' box (GTTAGGCGAAATATTA) between positions + 150 and + 166. Transfection of HeLa cells with this gene revealed that it is functioning in vivo and can produce U11 RNA.

Cystic fibrosis (CF) is one of the most common recessive conditions among whites, with an estimated carrier frequency of 1 in 25 in the United States. Population-based CF carrier screening was implemented in the United States in 2001. The number of mutations screened by each laboratory may vary; however, the 23 most common CF mutations recommended for screening by the American College of Medical Genetics and American College of Obstetricians and Gynecologists are included in all platforms. The c.1364C>A (p.A455E) mutation located in exon 10 of the CFTR gene is one of the 23 mutations. Because CFTR exon 10 and its flanking intronic regions are duplicated and transposed onto several other chromosomes of the human genome during evolution and function as unprocessed pseudogenes, variations in the CFTR pseudogenes may confound CF screening results for mutations located in exon 10 of the CFTR gene. We report an incorrectly identified carrier status for the c.1364C>A (p.A455E) mutation in a healthy individual using the Hologic InPlex CF assay. Further analysis revealed that the mutation resides in one of the CFTR pseudogenes. Because most commercial kits and laboratory-developed tests for CF carrier screening involve a short amplicon encompassing this mutation, this finding suggests that individuals with the c.1364C>A (p.A455E) mutation may require further investigation to avoid a false assignment of CF carrier status.

The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non-celiac gluten sensitivity, two pathologies affecting up to 6–8% of the human population worldwide. The wheat α-gliadin proteins contain three major CD immunogenic peptides: p31–43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-α3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of α-gliadin genes from diploid and polyploid wheat provided six types of α-gliadins (named 1–6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31–43 peptide were found in most of the α-gliadins. Variants of the DQ2.5-glia-α3 epitope were associated with specific types of α-gliadins. Remarkably, only type 1 α-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 α-gliadins seem to be the ancestral type as they are found in most of the α-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α-gliadins containing only immunogenic peptides., The Spanish Ministry of Economy and Competitiveness (Project AGL2013-48946-C3-1-R), the European Regional Development Fund (FEDER), and Junta de Andalucía (Project P11-AGR-7920) supported this work.

Main conclusion The complete chloroplast genome of two colchicine medicinal plants is reported for the first time. Deletion ofycf15 gene occurred only inColchicumbut not inGloriosaand suggests this as a potential marker for delineating the two species. Colchicum autumnale L. and Gloriosa superba L. are well-known sources of colchicine, a type of alkaloid and an ancient anti-inflammatory drug used to prevent gout. Accordingly, this alkaloid has been used as a chemical marker for identifying the expanded Colchicaceae family. In the present study, we report the complete chloroplast genome (cpDNA) sequence of two colchicine medicinal plants (G. superba and C. autumnale) that belong to the tribe Colchiceae of the Colchicaceae family. In C. autumnale, the circular double-stranded cpDNA sequence of 156,462 bp consists of two inverted repeat (IR) regions of 27,741 bp each, a large single-copy region (LSC) of 84,246 bp, and a small single-copy region (SSC) of 16,734 bp. The cpDNA sequence of G. superba is longer than that of C. autumnale (157,924 bp), which consists of two IRs (28,063 bp), an SSC (16,786 bp), and an LSC (85,012 bp). Significant structural differences between them were observed in the ycf15 gene. ycf15 gene was absent from C. autumnale cpDNA and affected the length of the chloroplast genome between the species. Furthermore, this gene loss event was specific to the expanded genus of Colchicum sensu Vinnersten and Manning. Therefore, this gene may be an effective and powerful molecular marker for identifying the Colchicum genus within the family.

Nanog plays an important role in maintaining the pluripotency of murine and human embryonic stem cells. However, the molecular features and transcriptional regulation of the NANOG gene have not been well investigated in pig. Here, we report, for the first time, that porcine NANOG is encoded by a single exon gene (SEG) mapped on chromosome 1 and has two daughter genes, one pseudogene NANOGP1 on chromosome 5 and one tandem duplicate on chromosome 1. The duplicated pseudogene NANOGP2 has high sequence similarity to NANOG, but does not encode a functional protein due to deletions and in-frame stop codons. The NANOGP1 contains four exons and three introns, but is short of the homeodomain sequence. Transcriptome analysis confirmed that NANOG mRNA in porcine iPS cells is transcribed from the SEG NANOG, but not from NANOGP1, because the NANOGP1 promoter is highly methylated, as confirmed by global DNA methylation analysis. The NANOG protein encoded by NANOG retains N, H, and C1/W/C2 domains. The H domain is required for nuclear translocation, while the C1/W/C2 domain ensures the NANOG regulatory function. Overexpression of NANOG in porcine embryonic fibroblasts promoted upregulation of its target genes SOX2, KLF4, and c-MYC. In conclusion, the functional porcine NANOG that is different in chromosomal structure from mouse and human genes is a single exon gene and encodes the functional NANOG protein that can be specifically regulated by OCT4/SOX2, and can promote the activation of target pluripotent factors in vivo.

In this study, we investigated the immunoglobulin heavy (IGH) gene locus of torafugu (Takifugu rubripes) from publicly available assembly sequences and presented an annotated locus map, including the IGHV genes, pseudogenes, and IGHC genes. Three new IGHV gene families (IGHV3-IGHV5) were discovered. We observed the interspersion of IGHV1 and IGHV2 family members and that they often intermingled with each other, while other family members were further interspersed. Conservation of the promoter and recombination signal sequences (RSS) was observed in a family-specific manner. In addition to known variable region genes present on chromosome 5 (current torafugu genome assembly), we found 34 additional IGHV genes on scaffold 287 and three novel potentially functional IGHD genes on scaffold 483. In total, the variable region of the torafugu IGH locus consists of at least 48 IGHV genes, seven IGHD genes, and six IGHJ genes. IGHC genes have also been mapped in this study, with three genes encoding immunoglobulin classes: IgT, IgM, and IgD. We confirmed the expression of newly identified IGHV3 family sequences in the spleen and kidney of adult torafugu and found a favorable IGHV segment usage by IgM and IgT. Possible structural variation in the IGHδ locus was observed based on the current torafugu assembly. The complete characterization of the torafugu IGH locus will facilitate detailed studies of large-scale mechanisms associated with the recombination of the variable region genes and will offer insights into the genetic basis of the potential diversity in the antibody response observed in torafugu.

In eukaryotes, the cyclin-dependent kinase Cdk1p (Cdc2p) plays a central role in entry into and progression through nuclear division during mitosis and meiosis. Cdk1p is activated during meiotic nuclear divisions by dephosphorylation of its tyrosine-15 residue. The phosphorylation status of this residue is largely determined by the Wee1p kinase and the Cdc25p phosphatase. In fission yeast, the forkhead-type transcription factor Mei4p is essential for entry into the first meiotic nuclear division. We recently identified cdc25(+) as an essential target of Mei4p in the control of entry into meiosis I. Here, we show that wee1(+) is another important target of Mei4p in the control of entry into meiosis I. Mei4p bound to the upstream region of wee1(+) in vivo and in vitro and inhibited expression of wee1(+), whereas Mei4p positively regulated expression of the adjacent pseudogene. Overexpression of Mei4p inhibited expression of wee1(+) and induced that of the pseudogene. Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene. In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene. These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.

Drosophila simulans is a close relative of the genetic model D. melanogaster. Its worldwide distribution in combination with the absence of segregating chromosomal inversions makes this species an increasingly attractive model to study the molecular signatures of adaptation in natural and experimental populations. In an effort to improve the genomic resources for D. simulans, we assembled and annotated the genome of a strain originating from Madagascar (M252), the ancestral range of D. simulans. The comparison of the M252 genome to other available D. simulans assemblies confirmed its high quality, but also highlighted genomic regions that are difficult to assemble with NGS data. The annotation of M252 provides a clear improvement with alternative splicing for 52% of the multiple-exon genes, UTRs for 70% of the genes, 225 novel genes and 781 pseudogenes being reported. We anticipate that the M252 genome will be a valuable resource for many research questions.

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.

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.

The common marmoset (Callithrix jacchus) is a New World monkey that is used frequently as a model for various human diseases. However, detailed knowledge about the MHC is still lacking. In this study, we sequenced and annotated a total of 854 kb of the common marmoset MHC region that corresponds to the HLA-A/G/F segment (Caja-G/F) between the Caja-G1 and RNF39 genes. The sequenced region contains 19 MHC class I genes, of which 14 are of the MHC-G (Caja-G) type, and 5 are of the MHC-F (Caja-F) type. Six putatively functional Caja-G and Caja-F genes (Caja-G1, Caja-G3, Caja-G7, Caja-G12, Caja-G13, and Caja-F4), 13 pseudogenes related either to Caja-G or Caja-F, three non-MHC genes (ZNRD1, PPPIR11, and RNF39), two miscRNA genes (ZNRD1-AS1 and HCG8), and one non-MHC pseudogene (ETF1P1) were identified. Phylogenetic analysis suggests segmental duplications of units consisting of basically five (four Caja-G and one Caja-F) MHC class I genes, with subsequent expansion/deletion of genes. A similar genomic organization of the Caja-G/F segment has not been observed in catarrhine primates, indicating that this genomic segment was formed in New World monkeys after the split of New World and Old World monkeys.

The incorporation of fragments of mitochondrial DNA (mtDNA) in the nuclear genome, known as numts (nuclear mitochondrial pseudogenes), undermines general assumptions concerning the use of mtDNA in phylogenetic and phylogeographic studies. Accidental amplifications of these nuclear copies instead of the mitochondrial target can lead to crucial misinterpretations, thus the correct identification of numts and their differentiation from true mitochondrial sequences are important in preventing this kind of error. Our goal was to describe the existence of cytochrome b (cytb) numts in the leafcutter ant Acromyrmex striatus (Roger, 1863). PCR products were directly sequenced using a pair of universal primers designed to amplify the cytb gene of these insects. Other species of leafcutter ants were also sequenced. The sequences were analyzed and the numts were identified by the presence of double peaks, indels and premature stop codons. Only A. striatus clearly showed the presence of numts, while the other species displayed the expected amplification of the mtDNA cytb gene target using the same primer pair. We hope that our report will highlight the benefits and challenges of using mtDNA in the molecular phylogenetic reconstruction and phylogeographic studies of ants, while establishing the importance of numts reports for future studies.

Increasing attention has been directed toward assessing mutational fallout of stereocilin (STRC), the gene underlying DFNB16. A major challenge is due to a closely linked pseudogene with 99.6% coding sequence identity. In 94 GJB2/GJB6-mutation negative individuals with non-syndromic sensorineural hearing loss (NSHL), we identified two homozygous and six heterozygous deletions, encompassing the STRC region by microarray and/or quantitative polymerase chain reaction (qPCR) analysis. To detect smaller mutations, we developed a Sanger sequencing method for pseudogene exclusion. Three heterozygous deletion carriers exhibited hemizygous mutations predicted as negatively impacting the protein. In 30 NSHL individuals without deletion, we detected one with compound heterozygous and two with heterozygous pathogenic mutations. Of 36 total patients undergoing STRC sequencing, two showed the c.3893A>G variant in conjunction with a heterozygous deletion or mutation and three exhibited the variant in a heterozygous state. Although this variant affects a highly conserved amino acid and is predicted as deleterious, comparable minor allele frequencies (MAFs) (around 10%) in NSHL individuals and controls and homozygous variant carriers without NSHL argue against its pathogenicity. Collectively, six (6%) of 94 NSHL individuals were diagnosed with homozygous or compound heterozygous mutations causing DFNB16 and five (5%) as heterozygous mutation carriers. Besides GJB2/GJB6 (DFNB1), STRC is a major contributor to congenital hearing impairment.

Flanders virus (FLAV) and Hart Park virus (HPV) are rhabdoviruses that circulate in mosquito-bird cycles in the eastern and western United States, respectively, and constitute the only two North American representatives of the Hart Park serogroup. Previously, it was suggested that FLAV is unique among the rhabdoviruses in that it contains two pseudogenes located between the P and M genes, while the cognate sequence for HPV has been lacking. Herein, we demonstrate that FLAV and HPV do not contain pseudogenes in this region, but encode three small functional proteins designated as U1, U2, and U3 that apparently arose by gene duplication. To further investigate the U1-U2-U3 region, we conducted the first large-scale evolutionary analysis of a member of the Hart Park serogroup by analyzing over 100 spatially and temporally distinct FLAV isolates. Our phylogeographic analysis demonstrates that although FLAV appears to be slowly evolving, phylogenetically divergent lineages co-circulate sympatrically.

A pseudogene, paralogous to rDNA 5.8S and ITS-2, is described in Meccus dimidiata dimidiata, M. d. capitata, M. d. maculippenis, M. d. hegneri, M. sp. aff. dimidiata, M. p. phyllosoma, M. p. longipennis, M. p. pallidipennis, M. p. picturata, M. p. mazzottii, Triatoma mexicana, Triatoma nitida and Triatoma sanguisuga, covering North America, Central America and northern South America. Such a nuclear rDNA pseudogene is very rare. In the 5.8S gene, criteria for pseudogene identification included length variability, lower GC content, mutations regarding the functional uniform sequence, and relatively high base substitutions in evolutionary conserved sites. At ITS-2 level, criteria were the shorter sequence and large proportion of insertions and deletions (indels). Pseudogenic 5.8S and ITS-2 secondary structures were different from the functional foldings, different one another, showing less negative values for minimum free energy (mfe) and centroid predictions, and lower fit between mfe, partition function, and centroid structures. A complete characterization indicated a processed pseudogenic unit of the ghost type, escaping from rDNA concerted evolution and with functionality subject to constraints instead of evolving free by neutral drift. Despite a high indel number, low mutation number and an evolutionary rate similar to the functional ITS-2, that pseudogene distinguishes different taxa and furnishes coherent phylogenetic topologies with resolution similar to the functional ITS-2. The discovery of a pseudogene in many phylogenetically related species is unique in animals and allowed for an estimation of its palaeobiogeographical origin based on molecular clock data, inheritance pathways, evolutionary rate and pattern, and geographical spread. Additional to the technical risk to be considered henceforth, this relict pseudogene, designated as "ps(5.8S+ITS-2)", proves to be a valuable marker for specimen classification, phylogenetic analyses, and systematic/taxonomic studies. It opens a new research field, Chagas disease epidemiology and control included, given its potential relationships with triatomine fitness, behaviour and adaptability.

The ecological model, Daphnia pulex (Cladocera: Daphniidae), is broadly distributed in Holarctic freshwater habitats and has been the subject of multidisciplinary study for over half a century, but never has a natural RNA virus infection been reported in daphnids. Here we report on a group of paleoviruses related to RNA dependent RNA polymerase in the genome of D. pulex. Phylogenetic analysis suggests that these paleoviruses are derived from a viral lineage within the genus Phlebovirus. Comparison of the genomic sequences flanking individual paleoviruses reveal that some are orthologous viral insertions having been present in the common ancestor of the D. pulex species complex, which is millions of years old. Still, we detected some sites that have the signature of purifying selection. In contrast, other paleoviruses in this group seem to be unique to specific host lineages and even contain undisrupted open reading frames, suggesting either more recent acquisition, or selective maintenance.

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.

Pancreatic ribonuclease gene (RNASE1) was previously shown to have undergone duplication and adaptive evolution related to digestive efficiency in several mammalian groups that have evolved foregut fermentation, including ruminants and some primates. RNASE1 gene duplications thought to be linked to diet have also been recorded in some carnivores. Of all mammals, bats have evolved the most diverse dietary specializations, mainly including frugivory and insectivory. Here we cloned, sequenced and analyzed RNASE1 gene sequences from a range of bat species to determine whether their dietary adaptation is mirrored by molecular adaptation. We found that seven insect-eating members of the families Vespertilionidae and Molossidae possessed two or more duplicates, and we also detected three pseudogenes. Reconstructed RNASE1 gene trees based on both Bayesian and maximum likelihood methods supported independent duplication events in these two families. Selection tests revealed that RNASE1 gene duplicates have undergone episodes of positive selection indicative of functional modification, and lineage-specific tests revealed strong adaptive evolution in the Tadarida β clade. However, unlike the RNASE1 duplicates that function in digestion in some mammals, the bat RNASE1 sequences were found to be characterized by relatively high isoelectric points, a feature previously suggested to promote defense against viruses via the breakdown of double-stranded RNA. Taken together, our findings point to an adaptive diversification of RNASE1 in these two bat families, although we find no clear evidence that this was driven by diet. Future experimental assays are needed to resolve the functions of these enzymes in bats.

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.

Members of the brachyuran crab family, Bythograeidae, are among the most abundant and common crabs in vent fields. However, their identification based on morphological characteristics often leads to incorrect species recognition due to a lack of taxonomic factors and the existence of sibling (or cryptic) species. For these reasons, we used DNA barcoding for vent crabs using mitochondrial cytochrome c oxidase subunit 1 (CO1). However, several nuclear mitochondrial pseudogenes (Numts) were amplified from Austinograea alayseae Guinot, 1990, using universal primers (Folmer primers). The Numts were characterized in six haplotypes, with 13.58-14.11% sequence divergence from A. alayseae, a higher nonsynonymous substitution ratio than true CO1, and the formation of an independent clade in bythograeids. In a neighbour-joining tree, the origin of the Numts would be expected to incorporate into the nucleus at an ancestral node of Austinograea, and they mutated more slowly in the nucleus than CO1 in the mitochondria. This evolutionary process may have resulted in the higher binding affinity of Numts for the Folmer primers than CO1. In the present study, we performed long PCR for the amplification of CO1 in A. alayseae. We also present evidence that Numts can introduce serious ambiguity into DNA barcoding, including overestimating the number of species in bythograeids. These results may help in conducting taxonomic studies using mitochondrial genes from organisms living in hydrothermal vent fields.

Phylogenetic analyses of nuclear and mitochondrial DNA sequences were used to test a recent phylogenetic hypothesis for the avian genus Gallicolumba that relied heavily on preserved museum study skins for DNA samples. Our comparisons show that several published gene sequences represent pseudogenes or chimeras of multiple taxa, and these sequences had an adverse influence on phylogeny estimation. A new phylogenetic hypothesis strongly supports the monophyly of Philippine bleeding-hearts, the monophyly of Gallicolumba sensu stricto, and places Leucosarcia as part of an Australasian radiation rather than sister to Alopecoenas. These findings clarify Gallicolumba biogeography and also highlight the need for greater caution in the use of suboptimal source material in molecular systematic studies.

UNLABELLED PREMISE OF THE STUDY The previously sequenced plastome of the hornwort Anthoceros angustus differs from that of other bryophytes by an expanded inverted repeat (IR) and the presence of a type I intron in the 23S ribosomal RNA (rrn23) gene. We assembled the plastome of the hornwort Nothoceros aenigmaticus, contrasted its architecture to that of other bryophytes, and assessed the phylogenetic significance of genomic characters in hornwort evolution. • METHODS The Nothoceros plastome was reconstructed from shotgun sequencing of genomic DNA. Comparison with the Anthoceros plastome revealed three structural differences. We sequenced these regions in taxa spanning the hornwort phylogeny. • KEY RESULTS The Nothoceros plastome is colinear with other bryophyte plastomes, but differs from the Anthoceros plastome by several gene regions located within the IR in Anthoceros being in the large single-copy region in Nothoceros, by the rrn23 gene lacking an intron, and by the rpl2 being a pseudogene. Comparisons across the hornwort phylogeny indicate that the first two characters are restricted to Anthocerotaceae, while rpl2 pseudogenization diagnoses the sister lineage to Anthocerotaceae. • CONCLUSIONS The Nothoceros plastome is structurally similar to that of most bryophytes. However, we identified more structural differences within hornworts than have been described within either the mosses or the liverworts. The distribution of the gene duplication involving the IR and an intron in the rrn23 gene are restricted to Anthocerotaceae. Occurrence of the intron and the conserved intron sequence between Anthoceros and distantly related chlorophyte algae may be due to horizontal gene transfer.

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.

Processed copies of genes generally evolve in neutral mode as pseudogenes, however, some of them might be important sources of new functional genes. The psiPGK1 pseudogene has been discovered in Schrenck salamander (Salamandrella schrenckii, Amphibia, Caudata, Hynobiidae) via polymerase chain reaction used to amplify the phosphoglycerate kinase 1 gene (PGK1). This pseudogene is an intronless copy of PGK1 gene absent of exon 6. Analysis of psiPGK1 pseudogene polymorphism has demonstrated that it lacks mutations, which results in shifts in the stop codons and reading frames, as well as that the interspecies variation of this pseudogene was inconsistent with the neutral model of evolution. In addition, the pattern of phylogeographic differentiation of the psiPGK1 variants mainly coincides with that observed in mitochondrial DNA. These observations allow it to be suggested that the psiPGK1 pseudogene is a new functional gene in the Schrenck salamander.

A number of studies require the determination of the genetic sex of mouse embryos before sexual differentiation and/or of mutant mice that display partial or complete sex reversal. The majority of current methods for sexing by PCR involve multiplexing of 2 primer pairs. We have developed a novel sexing PCR using a single primer pair that amplifies fragments from the X and the Y chromosome with a clear size difference between the respective amplicons. This assay provides a rapid and reliable method to identify the genetic sex of mice across different mouse strains.

Deficiency caused by mutations in the creatine transporter gene (SLC6A8/CT1) is an X-linked form of intellectual disability. The presence of highly homologous pseudogenes and high GC content of SLC6A8 genomic sequence complicates the molecular diagnosis of this disorder. To minimize the pseudogene interference, exons 2 to 13 of SLC6A8 were amplified as a single PCR product using gene-specific long-range PCR (LR-PCR) primers. The GC-rich exon 1 and its flanking intronic sequences were amplified separately in a short fragment under GC-rich conditions and a touchdown PCR program. Traditional Sanger sequence analysis of all coding exons of SLC6A8 from a 3-year-old boy with creatine transporter deficiency did not detect deleterious mutations. The long-range PCR product was used as template followed by massively parallel sequencing (MPS) on HiSeq2000. We were able to detect a tandem duplication involving part of exons 11 and 12 in the SLC6A8 gene. The deduced c.1592_1639dup133 mutation was confirmed to be a hemizygous insertion by targeted genomic DNA and cDNA Sanger sequencing. Combination of deep sequencing technology with long-range PCR revealed a novel intragenic duplication in the SLC6A8 gene, providing a definitive molecular diagnosis of creatine transporter deficiency in a male patient. (C) 2013 Elsevier Inc. All rights reserved

The tripartite motif protein (TRIM)5α/CypA fusion protein TRIMCyp in Old World monkeys is generally considered unable to restrict HIV-1 replication. Monkeys with TRIMCyp can serve as a unique animal model for studies of HIV-1 infection. The present study investigated the distribution and expression status of TRIMCyp in four species of macaques originating from China and its borderlands: pigtail macaques (Macaca nemestrina), rhesus macaques (Macaca mulatta), long-tailed macaques (Macaca fascicularis), and Tibetan macaques (Macaca thibetana). The results revealed that the frequencies of the TRIMCyp genotype were significantly different among different species and even within different populations of the same species. Interestingly, the TRIMCyp genotype was more prevalent among macaques originating from Yunnan and surrounding regions than those from other regions of China. Importantly, TRIMCyp individuals were first identified in Chinese M. mulatta originating from Yunnan, although multiple earlier studies failed to find CypA retrotransposition in this subspecies. Furthermore, TRIMe7-CypA, one of the splicing isoforms of the TRIMCyp transcript was expressed in M. nemestrina and M. mulatta but not M. fascicularis. The intra- and interspecies polymorphisms in the deduced TRIMCyp amino acid sequences of these macaques were also analyzed. Taken together, the data in this study provide important information about the genomic background of TRIMCyp among major species of Chinese macaques.

To date two closely related interleukin-1β genes (IL-1β1 and IL-β2) have been found in salmonids. The cloning of trout and salmon IL-1β3, and a salmon IL-1β4 pseudogene reveals that two types of IL-1β genes exist in teleost species. Type I teleost IL-1β genes, including salmonid IL-1β3, share a similar 6 coding exon structure as in tetrapods. Type II teleost IL-1β genes, e.g. salmonid IL-1β1-2, lack one or two coding exons at their 5'-end, and share higher identities within this subgroup than within the type I subgroup. Both types of IL-1β genes have been found in species of Salmoniformes, Perciformes and Beloniformes, suggesting the divergence occurred early in teleost evolution. Trout IL-1β3 is highly expressed in ovary suggesting a role in reproduction. A relatively high constitutive expression in gills, spleen and kidney and the up-regulation by PAMPs, proinflammatory cytokines and viral infection suggests IL-1β3 also has a role in inflammation and host defence.

Characterization and population genetic analysis of multilocus genes, such as those found in the major histocompatibility complex (MHC) is challenging in nonmodel vertebrates. The traditional method of extensive cloning and Sanger sequencing is costly and time-intensive and indirect methods of assessment often underestimate total variation. Here, we explored the suitability of 454 pyrosequencing for characterizing multilocus genes for use in population genetic studies. We compared two sample tagging protocols and two bioinformatic procedures for 454 sequencing through characterization of a 185-bp fragment of MHC DRB exon 2 in wolverines (Gulo gulo) and further compared the results with those from cloning and Sanger sequencing. We found 10 putative DRB alleles in the 88 individuals screened with between two and four alleles per individual, suggesting amplification of a duplicated DRB gene. In addition to the putative alleles, all individuals possessed an easily identifiable pseudogene. In our system, sequence variants with a frequency below 6% in an individual sample were usually artefacts. However, we found that sample preparation and data processing procedures can greatly affect variant frequencies in addition to the complexity of the multilocus system. Therefore, we recommend determining a per-amplicon-variant frequency threshold for each unique system. The extremely deep coverage obtained in our study (approximately 5000×) coupled with the semi-quantitative nature of pyrosequencing enabled us to assign all putative alleles to the two DRB loci, which is generally not possible using traditional methods. Our method of obtaining locus-specific MHC genotypes will enhance population genetic analyses and studies on disease susceptibility in nonmodel wildlife species.

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.

Human U1 small nuclear (sn)RNA, required for splicing of pre-mRNA, is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes, also located on chromosome 1 (1q12-21), were thought to be pseudogenes. However, many of these “variant” (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells, we have identified global transcriptome changes following interrogation of the Affymetrix Human Exon ST 1.0 array. Our results indicate that this vU1 snRNA regulates expression of a subset of target genes at the level of pre-mRNA processing. This is the first indication that variant U1 snRNAs have a biological function in vivo. Furthermore, some vU1 snRNAs are packaged into unique ribonucleoproteins (RNPs), and many vU1 snRNA genes are differentially expressed in human embryonic stem cells (hESCs) and HeLa cells, suggesting developmental control of RNA processing through expression of different sets of vU1 snRNPs.

Comparisons of the glandular kallikreins loci in human, mouse and rat revealed remarkable differences. For example, the mouse and the rat lack the genes encoding prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). In contrast, the intergenic region between KLK1 and KLK15 is devoid of genes and spans only 1.5 kb in humans, but encompasses 23 KLK1-like genes spanning 290 kb in the mouse. To further elucidate the evolution of glandular kallikrein genes, we investigated the structure and organization of these genes in the cotton-top tamarin (Saguinus oedipus), a New World monkey. We conclude that this species has no PSA gene. Moreover, the ortholog of the hK2 gene is a pseudogene, as it contains several mutations that preclude formation of a functional serine protease. The expression of this gene was probably silenced by a 15-bp deletion observed in an androgen response element in the upstream promoter region. Replacing the deleted base pairs in vitro with nucleotides from the human counterpart dramatically restored the transcriptional activity to a level that even surpassed that of the human ortholog. We also determined the nucleotide sequence of KLK15 and the intergenic region between this gene and KLK1 in the cotton-top tamarin. The region between KLK1 and KLK15 is conserved between the cotton-top tamarin and humans, and there are no signs of the extension seen in the mouse. KLK15 appeared to be functional, thus, we predict that it generates a protease with specificity similar to that of the human ortholog.

X chromosome inactivation is a mechanism of dosage compensation that regulates the expression of mammalian X-linked genes between XY males and XX females. This phenomenon is cis-acting, clonally heritable, and requires the presence of an X inactivation center (XIC). In our attempts to characterize this phenomenon, we have focused on the physical organization of the human XIC localized to Xq13. From previous studies, we had determined that the candidate XIC interval contained two loci (DXS128 and XIST) and was bound by the breakpoints of two structurally abnormal inactivated X chromosomes, a t(X;14) and an idic(Xp). Here we present a refined mapping of the XIC-containing region using the breakpoint of a late replicating rearranged X (rea(X)), and the initial characterization of a set of 40 yeast artificial chromosomes (YACs) derived from the XIC-containing region. These YACs form a 2.6 Mb contig which completely covers the XIC, and physically links the RPS4X, PHKA1, XIST, and DXS128E genes, as well as a laminin receptor pseudogene (LAMRP4). Furthermore, we have determined the relative orientations of these four genes, and have derived a restriction map of the region using the rare cutter enzymes BssHII, EagI, MluI, NruI, SalI, SfiI, SstII (or SacII), and NotI. We have identified at least 9 CpG-rich islands within this region, and have discovered a large (approximately 125 kb) inverted duplication proximal to the XIC based on symmetrical restriction patterns and homologous probes. We estimate the maximum size of the XIC-containing interval to be between 680 kb and 1200 kb, based on the localization of the breakpoints of the rearranged X chromosomes mentioned above. This lays the groundwork for the further characterization of the XIC region and the isolation of other expressed sequences therefrom.

African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei . The 26-megabase genome contains 9068 predicted genes, including ∼900 pseudogenes and ∼1700 T. brucei –specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi , and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major . Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

Transformer (tra) is the key gene that turns on the sex-determination cascade in Drosophila melanogaster and in some other insects. The honeybee Apis mellifera has two duplicates of tra, one of which (complementary sex determiner, csd) is the primary signal for complementary sex-determination (CSD), regulating the other duplicate (feminizer). Two tra duplicates have been found in some other hymenopteran species, resulting in the assumption that a single ancestral duplication of tra took place in the Hymenoptera. Here, we searched for tra homologues and pseudogenes in the Hymenoptera, focusing on five newly published hymenopteran genomes. We found three tra copies in the fig wasp Ceratosolen solmsi. Further evolutionary and expression analyses also showed that the two duplicates (Csoltra-B and Csoltra-C) are under positive selection, and have female-specific expression, suggesting possible sex-related functions. Moreover, Aculeata species exhibit many pseudogenes generated by lineage-specific duplications. We conclude that phylogenetic reconstruction and pseudogene screening provide novel evidence supporting the hypothesis of independent duplications rather an ancestral origin of multiple tra paralogues in the Hymenoptera. The case of C. solmsi is the first example of a non-CSD species with duplicated tra, contrary to the previous assumption that derived tra paralogues function as the CSD locus.

Ferritin, the major iron storage protein, performs its essential functions in the cytoplasm, nucleus and mitochondria. The variable assembly of 24 subunits of the Heavy (H) and Light (L) type composes the cytoplasmic molecule. In humans, two distinct genes code these subunits, both belonging to complex multigene families. Until now, one H gene has been identified with the coding sequence interrupted by three introns and more than 20 intronless copies widely dispersed on different chromosomes. Two of the intronless genes are actively transcribed in a tissue-specific manner. Herein, we report that FTH1P3, another intronless pseudogene, is transcribed. FTH1P3 transcript was detected in several cell lines and tissues, suggesting that its transcription is ubiquitary, as it happens for the parental ferritin H gene. Moreover, FTH1P3 expression is positively regulated during the cell differentiation process.

Nonallelic homologous recombination (NAHR) is one of the major mechanisms underlying copy number variation in the human genome. Although several disease-associated meiotic NAHR breakpoints have been analyzed in great detail, hotspots for mitotic NAHR are not well characterized. Type-2 NF1 microdeletions, which are predominantly of postzygotic origin, constitute a highly informative model with which to investigate the features of mitotic NAHR. Here, a custom-designed MLPA- and PCR-based approach was used to identify 23 novel NAHR-mediated type-2 NF1 deletions. Breakpoint analysis of these 23 type-2 deletions, together with 17 NAHR-mediated type-2 deletions identified previously, revealed that the breakpoints are nonuniformly distributed within the paralogous SUZ12 and SUZ12P sequences. Further, the analysis of this large group of type-2 deletions revealed breakpoint recurrence within short segments (ranging in size from 57 to 253-bp) as well as the existence of a novel NAHR hotspot of 1.9-kb (termed PRS4). This hotspot harbored 20% (8/40) of the type-2 deletion breakpoints and contains the 253-bp recurrent breakpoint region BR6 in which four independent type-2 deletion breakpoints were identified. Our findings indicate that a combination of an open chromatin conformation and short non-B DNA-forming repeats may predispose to recurrent mitotic NAHR events between SUZ12 and its pseudogene. Hum Mutat 33:15991609, 2012. (c) 2012 Wiley Periodicals, Inc.

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.

T-cell immunoglobulin domain and mucin domain containing protein 1 (TIM1), also known as a cellular receptor for hepatitis A virus (HAVCR1) or a molecule induced by ischemic injury in the kidney (KIM1), is involved in the regulation of immune responses. We investigated a natural selection history of TIM1 by comparative sequencing analysis in 24 different primates. It was found that TIM1 had become a pseudogene in multiple lineages of the New World monkey. We also investigated T cell lines originated from four different New World monkey species and confirmed that TIM1 was not expressed at the mRNA level. On the other hand, there were ten amino acid sites in the Ig domain of TIM1 in the other primates, which were suggested to be under positive natural selection. In addition, mucin domain of TIM1 was highly polymorphic in the Old World monkeys, which might be under balanced selection. These data suggested that TIM1 underwent a lineage-specific evolutionary pathway in the primates.

Telomerase is essential for proper functioning of telomeres in eukaryotes. We cloned and characterised genes for the protein subunit of telomerase (TERT) in the allotetraploid Nicotiana tabacum (tobacco) and its diploid progenitor species Nicotiana sylvestris and Nicotiana tomentosiformis with the aim of determining if allopolyploidy (hybridisation and genome duplication) influences TERT activity and divergence. Two of the three sequence variants present in the tobacco genome (NtTERT-C/s and NtTERT-D) revealed similarity to two sequence variants found in N. sylvestris and another variant (NtTERT-C/t) was similar to TERT of N. tomentosiformis. Variants of N. sylvestris origin showed less similarity to each other (80.5 % in the genomic region; 90.1 % in the coding sequence) than that between the NtTERT-C/s and NtTERT-C/t variants (93.6 and 97.2 %, respectively). The NtTERT-D variant was truncated at the 5' end, and indels indicated that it was a pseudogene. All tobacco variants were transcribed and alternatively spliced sequences were detected. Analysis of gene arrangements uncovered a novel exon in the N-terminal domain of TERT variants, a feature that is likely to be commonly found in Solanaceae species. In addition, species-specific duplications were observed within exon 5. The putative function, copy number and evolutionary origin of these NtTERT sequence variants are discussed.

The largest remaining carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is currently under threat of extinction due to a fatal contagious cancer-devil facial tumour disease. Low major histocompatibility complex (MHC) class I diversity is believed to have contributed to the transmission of the tumour allograft through devil populations. Here, we report low MHC class II variability in this species, with DA β chain genes (Saha-DAB1, 2 and 3) exhibiting very limited diversity and the sole α chain gene (Saha-DAA) monomorphic. Three, six and three alleles were found at Saha-DAB1, 2 and 3, respectively, with a predominant allele found at each locus. Heterozygosity at these three loci is low in the eastern population and modestly higher in northwestern individuals. The results are indicative of a selective sweep likely due to an infectious disease resulting in the fixation of selectively favoured alleles and depletion of genetic diversity at devil class II loci. Several attempts were made to isolate the other marsupial classical class II gene family, namely, DB, resulting in only one DBB pseudogene being found. These findings further support the view that this species has a compromised capacity to respond to pathogen evolution, emerging infectious diseases and environmental changes.