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Objective: This study endeavored to explore the relationship between exosome-derived lncRNA Double Homeobox A Pseudogene 8 (DUXAP8) and Chondroitin Polymerizing Factor 2 (CHPF2), and their roles in the pathogenesis of intracranial aneurysm (IA)., Methods: The shared targeted molecules (DUXAP8 and CHPF2) were detected via GSE122897 and GSE75436 datasets. A total of 312 patients with IAs were incorporated into this study. Exosomes were isolated from serum samples, and their identity was confirmed using Western blotting for exosomal markers (CD9, CD63 and ALIX). Inflammatory responses in IA tissues were evaluated using Hematoxylin-Eosin staining. CHPF2 protein concentration and the expression levels of DUXAP8 and CHPF2 mRNA in exosomal samples were assessed using Immunochemistry (IHC), Western Blotting, and qRT-PCR, respectively. Cell-based assays involving Human Umbilical Vein Endothelial Cells (HuvECs), including transfection with exosomal DUXAP8, Western Blotting, qRT-PCR, and Cell Counting Kit-8, were conducted. Receiver Operating Characteristic (ROC) curves were derived using SPSS., Results: DUXAP8 level affects the level of CHPF2. DUXAP8 expression within exosomes was associated with increased CD9, CD63, ALIX and CHPF2 levels during IA development and inflammatory stress. In HuvECs, transfection with exosomes carrying DUXAP8 siRNA resulted in reduced CHPF2 expression, whereas DUXAP8 mimic increased CHPF2 concentrations. The Area Under the ROC Curve (AUC) for exosomal DUXAP8 expression and CHPF2 levels, and aneurysm size was 0.768 (95% CI, 0.613 to 0.924), 0.937 (95% CI, 0.853 to 1.000), and 0.943 (95% CI, 0.860, 1.000), respectively., Conclusion: Exosome-derived DUXAP8 promotes IA progression by affecting CHPF2 expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract The CYP21A2 gene encoding 21‑hydroxylase is on chromosome 6p21.3 within the human leukocyte antigen (HLA) class III major histocompatibility complex and an association between congenital adrenal hyperplasia (CAH) due to 21‑hydroxylase deficiency and HLA class I and II alleles has been shown in genetically isolated populations. One-third of CAH causing alleles are 30-kb deletions due to homologous recombination events between active and pseudogenes resulting in chimeric genes. The aim of this study was to re-visit the association between the CYP21A2 variants and HLA polymorphisms in a large ethnically diverse cohort of patients with CAH who underwent comprehensive CYP21A2 genotyping, including specification of chimeric gene subtypes (CAH CH-1 through CH-9 of CYP21A1P/CYP21A2 chimeras; CAH-X CH-1 through CH-3 of TNXA/TNXB chimeras) in alleles with 30-kb deletions. The study population included 201 patients (86 males, 115 females, age 3–75 years) with CAH due to 21‑hydroxylase deficiency (159 classic, 42 nonclassic) and 194 parents. Based on the availability of parental genotype, we determined the haplotypes of CYP21A2 mutations and HLA types in 95 probands (190 alleles). Five prevalent haplotype associations were found: p.V281L and B*14-C*08 (P < 0.0001); p.I172N and DQB1*03 (P = 0.035); and of the chimeric genes caused by 30-kb deletions: CH-1 and A*03 (P = 0.033); CH-5 and C*06-DRB1*07 (P < 0.0001); and CAH-X CH-1 and DQB1*03 (P = 0.004). Our findings show that a number of associations between HLA alleles and haplotypes and CYP21A2 mutations, including large 30-kb deletions, exist commonly across ethnicities. These HLA associations may have clinical implications for patients with CAH and may provide insight into the genetics of this highly complex region of the human genome. Graphical abstract Unlabelled Image Highlights • CYP21A2 mutations are associated with HLA haplotypes, regardless of ethnicity. • p.V281L-HLA B*14-C*08 accounts for the majority of nonclassic alleles. • p.I172N-HLA DQB1*03 accounts for over one-third of simple virilizing alleles. • CYP21A1P/CYP21A2 and TNXA/TNXB chimeras are HLA linked. [ABSTRACT FROM AUTHOR]

Abstract The human genome contains an unusually large number of processed pseudogenes. The fact that processed pseudogenes are roughly 33% more abundant in our X chromosome than in our autosomes suggests that this overabundance is the result of the fact that human oogenesis is much longer than that of non-mammalian species. Here, we analyze the origins of the processed pseudogenes found on the human Y chromosome to determine whether human spermatogenesis also contribute to this overabundance. Our results show that human processed pseudogenes not only retrotranspose to the Y chromosome, but are also produced by genes on the Y chromosome. Furthermore, the fact that X chromosomes are three times more abundant than Y chromosomes likely explains why the euchromatic density of processed pseudogenes is three times higher in the X chromosome than in the Y chromosome. The large number of processed pseudogenes found in our genome is therefore due to the low substrate specificity of the L1 reverse transcriptase responsible for the reverse transcription of germline mRNA molecules into processed pseudogenes, as well as the life-long production of both male and female gametes. Highlights • Are the thousands of processed pseudogenes (PP) in mammalian genomes due to female meioses? • If so, mammalian spermatogenesis should not produce PP. • Here, we show that this prediction is wrong. • Human PP not only retrotranspose to the Y chromosome, but are also produced by genes on the Y chromosome. • The large number of PP found in the human genome is due to the life-long production of both male and female gametes. [ABSTRACT FROM AUTHOR]

Abstract Deoxynucleoside 5-monophosphate N -glycosidase, DNPH1 is a member of the nucleoside 2-deoxyribosyltransferase (NDT) family. This enzyme catalyzes the hydrolysis of deoxynucleoside monophosphates into free nucleobase moieties and 2-deoxyribose 5-phosphates. The DNPH1 enzymatic activity was first demonstrated in rats and then in human s. Subsequently the DNPH1 gene was identified in a variety of organisms, mainly in Metazoa. Herein, we demonstrate that despite DNPH1 genes being distributed in almost all metazoans, the occurrence of DNPH1genes is mosaic. For example, they cannot be found anywhere in the entire clade of Sauropsida or anywhere in the whole phyla of Arthropoda and Ctenophora. Even among mammals, there are organisms without functional DNPH1 protein (Camelidae and most likely Cetacea). By our knowledge, the DNPH1 gene is missing in plants, fungi and in majority of protists. Accordingly, the enzyme is apparently not of vital importance in all the branches of the Tree of Life. Surprisingly the DNPH1 gene may be found in archaea as well as in bacteria. This refers to the origin of the gene from the period before the archaea branched off from other bacteria. We show that the genomic and protein primary structures of DNPH1 are highly conserved and any modification in such a structure would result in conversion to a pseudogene, which could possibly be eliminated from the genome. Highlights • The DNPH1 gene structure has been conserved in all metazoans where present. • A majority of metazoans have a single functional DNPH1 gene. • The homolog of the DNPH1 gene is missing in many evolutionary branches. • DNPH1 is not a vital gene as many organisms can subsist without it. • DNPH1 in some archaea and bacteria is indistinguishable from that in Metazoa. [ABSTRACT FROM AUTHOR]

Abstract Highly conserved 18S rDNA sequences encode ribosomal RNA and evolve in a concerted manner. In this study, 178 sequences of 18S rDNA from the ridge-eyed flounder, Pleuronichthys cornutus , were analyzed. The total sequences yielded five distinguishable types of 18S rDNA—A, B, R, S, and L—that were defined based on sequence alignments, clone clustering, and recombination detection. The length of 176 clones ranged from 1838 bp to 1846 bp, with one particularly short clone reaching only 1466 bp and one long clone reaching up to1869 bp. As per current criteria for pseudogene inference, Type S was inferred to be a pseudogene due to its truncated length (380 bp) and low minimum free energy (−536.9 kcal·mol−1). Type L had a unique 20-bp insertion and was also predicted to be as a pseudogene. Types A and B showed 31 differential sites, which in Type A was consistent with 18S rDNA sequences found in six other flounders, while Type B and recombinant Type R were not. Maximum K2P genetic distances were calculated within Type B (0.0232) and R (0.0313); these were much higher than that of Type A (0.0093) or between the six flounders (0.011). Only Type A was detected by reverse transcription experiments and linked to functional ITS1 and 5.8S fragments; Types B and R were linked to both functional and pseudo genes. Accordingly, Type A is likely the functional gene, and Types B and R are probable pseudogenes. The heterogeneity of the five types revealed that 18S rDNA sequences evolved in a non-concerted evolution manner in P. cornutus. Furthermore, our results suggest that more features should be used to identify functional or pseudo genes. Highlights • The 18S rDNA in Pleuronichthys cornutus evolved in non-concerted evolution. • Five types of 18S rDNA were defined including length variation and recombination events. • Four more characteristics were suggested for rDNA pseudogene inference except the previous several criteria. [ABSTRACT FROM AUTHOR]

The rapid development of CRISPR technology is revolutionizing molecular approaches to the dissection of complex biological phenomena. Here we describe an alternative generally applicable implementation of the CRISPR-Cas9 system that allows for selective knockdown of extremely homologous genes. This strategy employs the lentiviral delivery of paired sgRNAs and nickase Cas9 (Cas9D10A) to achieve targeted deletion of splice junctions. This general strategy offers several advantages over standard single-guide exon-targeting CRISPR-Cas9 such as greatly reduced off-target effects, more restricted genomic editing, routine disruption of target gene mRNA expression and the ability to differentiate between closely related genes. Here we demonstrate the utility of this strategy by achieving selective knockdown of the highly homologous human genes FOXO3A and suspected pseudogene FOXO3B . We find the spJCRISPR strategy to efficiently and selectively disrupt FOXO3A and FOXO3B mRNA and protein expression; thus revealing that the human FOXO3B locus encodes a bona fide human gene. Unlike FOXO3A, we find the FOXO3B protein to be cytosolically localized in both the presence and absence of active Akt. The ability to selectively target and efficiently disrupt the expression of the closely-related FOXO3A and FOXO3B genes demonstrates the efficacy of the spJCRISPR approach. [ABSTRACT FROM AUTHOR]

One cluster of 5S rDNA per haploid genome is the most common pattern among Heteroptera. However, in Chariesterus armatus , highly scattered signals were noticed. We isolated and characterized the entire 5S rDNA unit of C . armatus aiming to a deeper knowledge of molecular organization of the 5S rDNA among Heteroptera and to understand possible causes and consequences of 5S rDNA chromosomal spreading. For a comparative analysis, we performed the same approach in Holymenia histrio with 5S rDNA restricted to one bivalent. Multiple 5S rDNA variants were observed in both species, though they were more variable in C . armatus , with some of variants corresponding to pseudogenes. These pseudogenes suggest birth-and-death mechanism, though homogenization was also observed (concerted evolution), indicating evolution through mixed model. Association between transposable elements and 5S rDNA was not observed, suggesting spreading of 5S rDNA through other mechanisms, like ectopic recombination. Scattered organization is a rare example for 5S rDNA, and such organization in C . armatus genome could have led to the high diversification of sequences favoring their pseudogenization. [ABSTRACT FROM AUTHOR]

A growing body of evidence has indicated that long non-coding RNAs (lncRNAs) function as competing endogenous RNAs (ceRNAs) during tumorigenesis. In this study, the qRT-PCR results revealed that the lncRNA ferritin heavy chain 1 pseudogene 3 (FTH1P3) was over-expressed in oral squamous cell carcinoma (OSCC) and decreased the survival rate of OSCC patients. Ectopic expression of FTH1P3 facilitates cell proliferation and colony formation in OSCC cells. Moreover, FTH1P3 acted as a competitive endogenous RNA (ceRNA), effectively becoming sponge for miR-224-5p and thereby modulating the expression of fizzled 5. Importantly, expression analysis revealed that both FTH1P3 and fizzled 5 were up-regulated in OSCC cell lines and tissues, and over-expression of fizzled 5 also functioned as an oncogene in OSCC cells. Our data demonstrated FTH1P3 facilitated OSCC progression by acting as a molecular sponge of miR-224-5p to modulate fizzled 5 expression. Thus, targeting the ceRNA network referring FTH1P3 may be a therapeutic target for treatment of OSCC. [ABSTRACT FROM AUTHOR]

Segmental duplication, or low-copy repeat (LCR) event, occurs during primate evolution and is an important source of genomic diversity, including gain or loss of gene function. The human chromosome 7q 11.23 is related to the William–Beuren syndrome and contains large region-specific LCRs composed of blocks A, B, and C that have different copy numbers in humans and different primates. We analyzed the structure of POM121 , NSUN5 , FKBP6 , and TRIM50 genes in the LCRs of block C. Based on computational analysis, POM121B created by a segmental duplication acquired a new exonic region, whereas NSUN5B ( NSUN5C ) showed structural variation by integration of HERV-K LTR after duplication from the original NSUN5 gene. The TRIM50 gene originally consists of seven exons, whereas the duplicated TRIM73 and TRIM74 genes present five exons because of homologous recombination-mediated deletion. In addition, independent duplication events of the FKBP6 gene generated two pseudogenes at different genomic locations. In summary, these clustered genes are created by segmental duplication, indicating that they show dynamic evolutionary events, leading to structure variation in the primate genome. [ABSTRACT FROM AUTHOR]

Green light irradiation facilitates the somatic growth of barfin flounder ( Verasper moseri ). However, the V. moseri visual system, which may be associated with somatic growth by acting on the endocrine system upon exposure to this particular wavelength, remains largely unexplored. Herein, we characterized the visual opsin repertoire of V. moseri to understand the molecular basis underlying this effect. The five types of visual opsins that are found in vertebrates were cloned from RNA that was extracted from the eyes of V. moseri . Notably, V. moseri possessed one pseudogene ( RH2-A ) and two intact ( RH2-B and RH2-C ) copies of “green-sensitive” opsin genes. The wavelengths of maximum absorption spectra (λ max ) for each of the reconstituted photopigments were 552 nm for “red-sensitive” LWS , 506 nm for RH2-B , 490 nm for RH2-C , 482 nm and 416 nm for “blue-sensitive” SWS2A and SWS2B , respectively, 367 nm for “ultraviolet-sensitive” SWS1 , and 494 nm for “dim-light sensitive rhodopsin” RH1 . The λ max of SWS2A was longer than that of any other reported vertebrate SWS2 opsin. By measuring the expression level of these opsin genes with quantitative RT-PCR in 3-, 15-, and 27-month-old fish, we found that RH2-B and SWS2A were expressed at a constant level, whereas the expression of LWS , RH2-C , SWS2B , and SWS1 opsin genes decreased, and that of RH1 increased with age. Barfin flounders inhabit inshore waters at a young age and expand their habitat to deep sea areas as they age, and green light is relatively abundant in deep water compared to the lights of other wavelengths in shallow water. Our results indicate that gene repertoire and expression profile of the opsin genes of barfin flounder are adaptive to their habitat shift that occurs during development, with some opsins acquiring a distinct λ max . [ABSTRACT FROM AUTHOR]

• The first mitochondrial genome of the carnivorous plant genus Genlisea. • G. tuberosa mitochondrial genome has 36 protein-coding genes, 40 tRNAs, and 4 rRNAs. • The genes are similar to those from other Lentibulariaceae, including the LAGLIDAD endonuclease. • Gene content and organization are similar to other sequenced MT from non-carnivorous species. Sequenced genomic data for carnivorous plants are scarce, especially regarding the mitogenomes (MTs) and further studies are crucial to obtain a better understanding of the topic. In this study, we sequenced and characterized the mitochondrial genome of the tuberous carnivorous plant Genlisea tuberosa, being the first of its genus to be sequenced. The genome comprises 729,765 bp, encoding 80 identified genes of which 36 are protein-coding, 40 tRNA, four rRNA genes, and three pseudogenes. An intronic region from the cox1 gene was identified that encodes an endonuclease enzyme that is present in the other sequenced species of Lentibulariaceae. Chloroplast genes (pseudogene and complete) inserted in the MT genome were identified, showing possible horizontal transfer between organelles. In addition, 50 pairs of long repeats from 94 to 274 bp are present, possibly playing an important role in the maintenance of the MT genome. Phylogenetic analysis carried out with 34 coding mitochondrial genes corroborated the positioning of the species listed here within the family. The molecular dynamism in the mitogenome (e.g. the loss or pseudogenization of genes, insertion of foreign genes, the long repeats as well as accumulated mutations) may be reflections of the carnivorous lifestyle where a significant part of cellular energy was shifted for the adaptation of leaves into traps molding the mitochondrial DNA. The sequence and annotation of G. tuberosa 's MT will be useful for further studies and serve as a model for evolutionary and taxonomic clarifications of the group as well as improving our comprehension of MT evolution. [ABSTRACT FROM AUTHOR]

Abstract: Aquatic organisms such as cichlids, coelacanths, seals, and cetaceans are active in UV–blue color environments, but many of them mysteriously lost their abilities to detect these colors. The loss of these functions is a consequence of the pseudogenization of their short wavelength-sensitive (SWS1) opsin genes without gene duplication. We show that the SWS1 gene (BdenS1ψ) of the deep-sea fish, pearleye (Benthalbella dentata), became a pseudogene in a similar fashion about 130million years ago (Mya) yet it is still transcribed. The rates of nucleotide substitution (~1.4×10−9/site/year) of the pseudogenes of these aquatic species as well as some prosimian and bat species are much smaller than the previous estimates for the globin and immunoglobulin pseudogenes. [Copyright &y& Elsevier]

Abstract: Extensive gene duplication arranged in a tandem array is rare in the plastome of embryophytes. Interestingly, we found pseudogene copies of the trnF gene in the genus Jaltomata, the sister genus of Solanum where such gene duplication has been previously reported. In each Jaltomata sequence available we found two pseudogene copies in close 5′-proximity to the original functional gene. The size of each pseudogene copy ranged between 17 and 48bp and the anticodon domain was identified as the most conserved element. A common ATT(G)n motif is particularly interesting and its modifications were found to border the 3′ of the duplicated regions. Other motifs were partial residues, or entire parts of the T- and D-domains, and both domains proved to be variable in length among the pseudogenes identified. The residues of the 3′ and 5′ acceptor stem were not found among the copies. We further compared the newly discovered copies of Jaltomata with those ones previously described from Solanum and inferred phylogenetic relationships of the copies aligned. The evolution of Solanum copies, in contrast to Jaltomata, is hard to explain as resulting only in parsimonious changes since reticulate evolutionary patterns were detected among the copies. The dynamic evolutionary patterns of Solanum might be explained by possible inter- or intrachromosomal recombination. [Copyright &y& Elsevier]

Abstract: Aspartic proteinases form a widely distributed protein superfamily, including cathepsin D, cathepsin E, pepsins, renin, BACE and napsin. Human napsin genes are located on human chromosome 19q13, which comprises napsin A and napsin B. Napsin B has been annotated as a pseudogene because it lacks an in-frame stop codon; its nascent chains are cotranslationally degraded. Until recently, there have been no studies concerning the molecular evolution of the napsin protein family in the human genome. In the present study, we investigated the evolution and gene organization of the napsin protein family. Napsin B orthologs are primarily distributed in primates, while napsin A orthologs are the only napsin genes in other species. The corresponding regions of napsin B in the available sequences from primate species contain an in-frame stop codon at a position equivalent to that of human napsin A. In addition, a rare single-nucleotide polymorphism (SNP) that creates a proper stop codon in human napsin B was identified using HapMap populations. Recombinant protein expression and three-dimensional comparative modeling revealed that napsin B exhibits residual activity toward synthetic aspartic protease substrates compared with napsin A, presumably through a napsin B-specific Arg287 residue. Thus, napsin B was duplicated from napsin A during the early stages of primate evolution, and the subsequent loss of napsin B function during primate evolution reflected ongoing human-specific napsin B pseudogenization. [Copyright &y& Elsevier]

Abstract: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease of steroid biosynthesis in humans. More than 90% of all CAH cases are caused by mutations of the 21-hydroxylase gene (CYP21A2), and approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. In this study, the CYP21A2 gene was genotyped in 50 patients in Tunisia with the clinical diagnosis of 21-hydroxylase deficiency. CYP21A2 mutations were identified in 87% of the alleles. The most common point mutation in our population was the pseudogene specific variant p.Q318X (26%). Three novel single nucleotide polymorphism (SNP) loci were identified in the CYP21A2 gene which seems to be specific for the Tunisian population. The overall concordance between genotype and phenotype was 98%. With this study the molecular basis of CAH has been characterized, providing useful results for clinicians in terms of prediction of disease severity, genetic and prenatal counseling. [Copyright &y& Elsevier]

Abstract: Pseudogenes, the ‘genomic fossils’ present portrayal of evolutionary history of human genome. The human genes configuring pseudogenes are also now coming forth as important resources in the study of human protein evolution. In this communication, we explored evolutionary conservation of the genes forming pseudogenes over the genes lacking any pseudogene and delving deeper, we probed an evolutionary rate difference between the disease genes in the two groups. We illustrated this differential evolutionary pattern by gene expressivity, number of regulatory miRNA targeting per gene, abundance of protein complex forming genes and lesser percentage of protein intrinsic disorderness. Furthermore, pseudogenes are observed to harbor sequence variations, over their entirety, those become degenerative disease-causing mutations though the disease involvement of their progenitors is still unexplored. Here, we unveiled an immense association of disease genes in the genes casting pseudogenes in human. We interpreted the issue by disease associated miRNA targeting, genes containing polymorphisms in miRNA target sites, abundance of genes having disease causing non-synonymous mutations, disease gene specific network properties, presence of genes having repeat regions, affluence of dosage sensitive genes and the presence of intrinsically unstructured protein regions. [Copyright &y& Elsevier]

Abstract: In the present study, we cloned and characterized the buffalo (Bubalus bubalis) OCT4 ortholog expressed in embryonic stem cell (ESC) like cells and its promoter region. The 5′- and 3′-RACE experiments were conducted to analyze the transcription initiation site and regulatory regions. The comparative analysis of buffalo OCT4 promoter with other mammalian orthologs revealed high conservation. Among the regulatory regions highest similarity was observed between buffalo, bovine and sheep. Interestingly, buffalo OCT4 promoter exhibited a 78bp deletion between two proximal enhancers (PE-1A and PE-1B) when compared to other mammalian orthologs. 5′-RACE revealed four different transcription start sites for OCT4 gene. As far as we know there is no previous report regarding multiple transcription initiation sites for OCT4 gene in any species. In addition, we identified expression of four pseudogenes in buffalo ESC-like cells. Among the multiple transcripts characterized, we found four cDNA clones (1083bp) derived from ESC-like cells sharing 96.9–99.3% sequence homology with the parent gene and having the capacity of encoding 139, 206, 206 and 324 amino acid long truncated proteins. Multiple pseudogenes have been proposed for OCT4 which might contribute to the false detection of this gene during expression studies. However, only few of them were reported to be transcribed and none were reported to be translated in stem cells. Western blot analysis of OCT4 protein using ESC-like cells revealed multiple bands, indicating that some of the hypothetical pseudogenes are being translated. These novel pseudogenes or their protein products may have some important regulatory functions. [Copyright &y& Elsevier]

• The current goat genome harbors 50 β-defensin genes. • Some duplicated β-defensin genes in goat chromosome 27 may be derived from gene copy number variation. • The annotation of sheep and cattle β-defensins appears to be incomplete in the genome. • Goat β-defensin 1 is the highest expressed β-defensins in the small intestine. Defensins represent a family of cysteine-rich peptides that have broad-spectrum antimicrobial activities and serve as a typical kind of effector molecule in the immunity. Ruminant species have a large number of β-defensins in the absence of α- and θ-defensins. It is well-known that the genomes of sheep and cattle harbor at least 43 and 57 β-defensin genes, respectively. However, the repertoire of the goat β-defensin gene family has not been fully elucidated. In this study, we identified a total of 50 β-defensins from the goat genome, including 48 functional genes and 2 pseudogenes. Cross-species genomic and evolutionary analyses showed that all of the β-defensins in goat chromosomes 8, 13 and 23 present one-to-one orthologous relationships to their sheep and cattle counterparts, whereas some β-defensin genes in goat chromosome 27 are goat-specific. Moreover, we observed that some duplicated genes in goat chromosome 27 may be derived from gene copy number variation, and the annotation of sheep and cattle β-defensins appears to be incomplete in the genome. Importantly, real-time PCR analysis showed that 17 β-defensins are expressed in the small intestine with abundant cBD1s expression. These findings significant increased our knowledge of ruminant β-defensin and provided useful information for genetic studies, as well as providing a foundation for future research exploring the role of defensins in the immune response. [ABSTRACT FROM AUTHOR]

Abstract: Prions represent a new class of infectious agents. The pathogenic prion protein (PrPSc) is known as the trigger of bovine transmissible spongiform encephalopathy (TSE). By contrast, an oral transmission of PrPSc and an ensuing infection seems to be blocked in non-ruminants such as pigs. Several investigations postulate that the ribosomal protein SA (RPSA) previously named 37-kDa laminin receptor precursor (LRP)/67-kDa laminin receptor (LR) is the candidate for binding and internalization of externally added cellular prion protein in the gut. We isolated a porcine ribosomal protein SA cDNA that consists of 1064 bp with an open reading frame of 885 bp encoding a 295 aa protein. The alignment of vertebrate ribosomal protein SA sequences displayed interspecies differences between cattle and pigs at positions 241 and 272 in the putative indirect PrP interaction site (aa 180–285) on RPSA. A PAC library screen revealed the existence of two processed ribosomal protein SA pseudogenes (RPSAP1 and RPSAP3) and of one non-processed pseudogene (RPSAP2). The pseudogenes have been assigned to SSC6 and SSC1 by hybrid panel analyses and FISH. Compared with the porcine cDNA 3, 7, and 13 insdels, 36, 25, and 57 single nucleotide exchanges and 6, 10, and 8 premature stop codons have been deciphered for RPSAP1, RPSAP2, and RPSAP3. In the 5′, 3′, and intron like regions, 2 (RPSAP1), 10 (RPSAP2), and 4 (RPSAP3) repeats have been detected. Basically, the repeats belong to one of the class/family LINE/L1, SINE/tRNA-Glu and DNA/MER1_type. We conclude that the pig genome contains multiple copies of the RPSA sequence probably as a consequence to maintain the multifunctionality of the mature protein. [Copyright &y& Elsevier]

Abstract: Genome reduction, a typical feature of symbiotic bacteria, was analyzed in the last stages of evolution of Buchnera aphidicola, the primary aphid endosymbiont, in two neutrally evolving regions: the pseudogene cmk and an intergenic region. These two regions were examined in endosymbionts from several lineages of their aphid host Rhopalosiphum padi, and different species of the same genus, whose divergence times ranged from 0.62 to 19.51 million years. Estimates of nucleotide substitution rates were between 4.3 and 6.7×10−9 substitution/site/year, with G or C nucleotides being substituted around four times more frequently than A or T. Two different types of indel events were detected, of which many were small (1–10 nt) but one was large (about 200 nucleotides).With respect to the large one and considering the proportion and size of the deletions and insertions, the reduction rate was 1.3×10−8 lost nucleotides/site/year. We propose a stepwise scenario for the last stages of evolution in B. aphidicola: together with a very slow and gradual degradation, considerable indels would punctually emerge. The only restriction to large deletion fixation is that the lost fragment does not contain essential genes. [Copyright &y& Elsevier]

Abstract: The Atlantic salmon (Salmo salar) has been widely used as a model species in studies of olfactory signal transduction and processing. Here we report the isolation and characterisation of salmon olfactory receptor (SOR) and salmon vomeronasal receptor (SVR) partial sequences from Atlantic salmon. Six groups of SOR sequences (SORA–F) and three groups of SVR sequences (SVRA–C) were identified. All SORB, SORF, SVRB and SVRC sequences contained uninterrupted open reading frames. However, all SORA sequences and members of the SVRA sequence family contained multiple stop codons while SORC and SORE sequences were truncated in the 3′ region of the sequence. Full length SORF and almost complete SORB sequences displayed amino acid residues and motifs conserved in fish olfactory receptor genes. In sequence phylogenies, SOR sequences fell into the main olfactory receptor (MOR) type I clade and were most closely related to either δ or ζ reference sequences, while all SVR sequences grouped within a clade of fish type 2 vomeronasal receptor (V2R) sequences. A family of sequences (Sasa CaSR1–6), isolated using the same degenerate primers that amplified SVR sequences, clustered within a group of calcium sensing receptor (CaSR) sequences. Analysis of tissue expression patterns of sequences by reverse transcriptase polymerase chain reaction showed that they were transcribed in olfactory epithelium (SORB, SORF, all SVR and Sasa CaSR sequences), testis (SORB, SORD and Sasa CaSR) and/or anterior kidney (SORB and Sasa CaSR). Similar analysis of expression supported the identification of SORA sequences as non-transcribed pseudogene(s). Although the level of occurrence of OR pseudogenes is within the range found for other, well-characterised vertebrate OR genomes, it does not seem to reflect the importance of olfaction in the biology of the Atlantic salmon. [Copyright &y& Elsevier]

Abstract: Characterization of the Gallus gallus α-amylase gene family revealed that the chicken genome contains two distinct amy loci. One of the two loci is expressed in the chicken pancreas while cDNA clones for the second locus were detected in a library constructed from liver mRNA. Fluorescent in situ hybridization to chromosome spreads showed that the two loci are both located on chromosome 8 within the chicken genome. Moreover, each locus contains both an intact, expressed gene copy as well as a pseudogene. The expressed gene and the pseudogene are arranged in a divergent configuration in the pancreatic amy locus, while in the hepatic locus the intact gene and the pseudogene are arranged in tandem. The data suggest a complex pattern of evolution for the chicken amylase gene family which includes multiple gene duplication events, insertion/deletion events, as well as changes in spatial expression patterns. [Copyright &y& Elsevier]

Abstract: We describe the complete opsin gene families from the sequenced fugu and Tetraodon pufferfish genomes. We report the convergent loss of function of an anciently duplicated, functionally divergent RH2 or “green-sensitive” opsin gene in both pufferfish lineages, designated RH2-2. In fugu, RH2-2 apparently ceased to function very recently following a transposon-induced deletion that truncated the N-terminal 115 amino acids from the translated protein. Although a lack of frameshift or nonsense mutations in the fugu RH2-2 pseudogene suggests that the gene was lost very recently in this lineage, we were unable to detect any evidence of a selective sweep associated with the fixation of the truncated allele from population data. Interspecific comparison of the remaining fugu RH2-2 coding sequence paradoxically indicates that the gene was under strong purifying selection until the truncation occurred. [Copyright &y& Elsevier]

There is increasing evidence that DNA duplication is a common and ongoing process that plays a major role in molecular evolution of genomes and that a large fraction of the duplicated gene copies becomes non-functional by accumulation of deleterious mutations. In order to describe this phenomenon, we systematically searched the 6404 intergenic regions (IRs) of the genome of Saccharomyces cerevisiae for traces of coding sequences presenting degenerated but still recognizable sequence similarity with active open reading frames (5823 annotated ORFs). We detected a total of 124 anciently coding regions, or “gene relics”, showing similarity to a total of 149 distinct active ORFs. This set of relics shows a continuum of sequence degeneration from those whose sequence is slightly altered compared to the functional ORF (classically defined as pseudogenes), to those that contains so many deleterious mutations, as to reach the limit of recognition. Gene relics are more concentrated in the subtelomeric regions of the chromosomes, reflecting the high plasticity of these regions. The presence of relics also revealed ancestral duplication events of chromosomal segments that were previously undetected. Some of these segments are intermingled with the more easily recognizable ancestral blocks of duplication, indicating successive duplication events. We present a compilation of all the data available, leading to a total of 278 pseudogenes in the genome of S. cerevisiae. [Copyright &y& Elsevier]

Numerous theories have been proposed to account for the pronounced differences in the quantity of non-coding DNA among eukaryotic genomes, but the current repertoire remains incomplete because the only explicit mechanisms it provides involve DNA gain. It has been proposed more recently that biases in spontaneous insertions and deletions (indels) can lead to genome shrinkage by mutational mechanisms alone. The present article provides the first detailed critical discussion of this approach, and covers three different ideas related to it: (1) the general notion of DNA loss by deletion bias, (2) the “DNA loss hypothesis” which supposes that variation in genome size can be attributed to differences in DNA loss rate, and (3) the “mutational equilibrium model” which attempts to describe the long-term evolution of genome size. The mutational equilibrium model is found to be problematic, and it is noted that DNA loss by small indels is too slow in real time to determine variation in genome size above a relatively low threshold. Some alternative explanations for the observed patterns are provided, and the critique also identifies some potential problems with the current dataset. These include a failure to cite a more detailed (and somewhat contradictory) mammalian dataset, a questionable use of arithmetic means with highly skewed data, and important discrepancies among the particular DNA sequences so far analyzed. Overall, evolutionary reductions in genome size are considered important, but the specific mechanism relating to small deletion bias is far too weak to be accepted as a primary determinant of genome size variation in general. [Copyright &y& Elsevier]

Using a computational approach, we have identified 49 cytochrome c (cyc) pseudogenes in the human genome. Analysis of these provides a detailed description of the molecular evolution of the cyc gene. Almost all of the pseudogenes are full-length, and we have concluded that they mostly originated from independent retrotransposition events (i.e. they are processed). Based on phylogenetic analysis and detailed sequence comparison, we have further divided these pseudogenes into two groups. The first, consisting of four young pseudogenes that were dated to be between 27 and 34 Myr old, originated from a gene almost identical to the modern human cyc gene. The second group of pseudogenes is much older and appears to have descended from ancient genes similar to modern rodent cyc genes. Thus, our results support the observation that accelerated evolution in cyc sequence had occurred in the primate lineage. The oldest pseudogene in the second group, dated to be over 80 Myr old, resembles the testis-specific cyc gene in modern rodents. It is likely that the mammalian ancestor had both the somatic and the testis-specific cyc genes. While the testis-specific gene is still functional in modern rodents, the human has lost it, retaining only a pseudogene in its place. Thus, our study may have identified a pseudogene that is a dead relic of a gene that has completely died off in the human lineage. [Copyright &y& Elsevier]

• Four groups of olfactory receptor genes in common carp were identified by mining all existing genomic resources. • Selection pressure of olfactory receptor in common carp after WGD were relaxed. • Except ORAs, most olfactory genes only expressed after gastrula stage and in general, few olfactory receptors expressed before hatching. The environment contains a large extent of chemical information, which could be detected as olfactory sense. Olfactory in vertebrates plays important roles on many aspects during life time, including localizing prey or food, avoiding predators, mating behavior and social communication. Considering the essential role of olfactory receptors in the specific recognition of diverse stimuli, understanding the evolutionary dynamics of olfactory receptors in teleost means a lot, especially in the allotetraploid common carp, who has undergone the fourth whole-genome duplication event. Here, we identified the whole set of olfactory receptor genes in representative teleosts and found a significant contraction in common carp when compared with other teleosts. Odorant receptor genes (OR) occupy the most among four groups of olfactory receptors, including 33 functional genes and 16 pseudogenes. Furthermore, 6 trace amine-associated receptor (TAAR) genes (including 1 pseudogene), 7 odorant-related-A receptor genes, and 10 olfactory C family receptor genes (including 3 pseudogenes) were identified in common carp. Phylogenetic and motif analysis were performed to illustrate the phylogenetic relationship and structural conservation of teleost olfactory receptors. Selection pressure analysis suggested that olfactory receptor groups in common carp were all under relaxed purifying-selection. Additionally, gene expression divergences for olfactory receptor genes were investigated during embryonic development stages of common carp. We aim to determine the abundance of common carp olfactory receptor genes, explore the evolutionary fate and expression dynamics, and provide some genomic clues for the evolution of polyploid olfactory after whole-genome duplication and for future studies of teleost olfactory. [ABSTRACT FROM AUTHOR]

• B2M gene has no pseudogene and ideal for normalization of gene expression data. • B2M with no pseudogenes and ACTB and GAPDH with pseudogenes were validated by PCR. • Phylogeny reveals the evolutionary conservation of pseudogenes among mammals. • ValidPrime Assay is useful to nullify the signal from pseudogene amplification. Pseudogenes are duplicated or retrotransposed DNA sequences of native functional genes. Amplification of pseudogenes along with gene of interest often produces false positive results, which is an innate problem in Reverse Transcription-quantitative PCR (RT-qPCR). Selecting a reference gene without any interference from pseudogene amplification is therefore a challenge to overcome. Among the common reference genes used for normalization (ACTB, GAPDH , HPRT1 , TUBB , RNA18SN1 and B2M), B2M was found to have no pseudogenes in silico , which has also been confirmed by PCR and RT-qPCR. We also assessed the effect of pseudogenes on the determination of the stability of reference genes through data mining. The phylogenetic analysis of pseudogenes and functional genes revealed high sequence similarity among mammals. In addition, we demonstrated the deduction of pseudogene amplification signal using ValidPrime Assay (VPA) under conditions where genomic DNA contamination could not be avoided. Hence, we recommend the use of pseudo-free reference gene with consistent expression in the samples of interest or use VPA normalization method, where genomic DNA or pseudogene amplification is inevitable. [ABSTRACT FROM AUTHOR]

• Multi-dimensional -omics data can be used to study various functional aspects of the genome. • Using -omic data, expression signals in the pseudogene regions in M. smegmatis were investigated • Further analysis showed sequencing errors in many potentially important pseudogenic regions. Large-scale -omics data now allows for investigating genome-wide functional elements. Using RNA-Seq data, we tested the expression of 134 pseudogenes which do not show duplicated protein coding genes in the M. smegmatis genome. We observe significant expression and translation of 28 pseudogenes. Further examination using RNA-Seq reads suggested the sequencing errors in many pseudogenes. These include some of the functionally relevant genes such as recN and manB. We propose that the analysis of transcriptional and translational landscape using multi-dimensional -omics data could shed light on the current annotations of the bacterial pseudogenes. [ABSTRACT FROM AUTHOR]

• Plastomes of two edible Allium species of subg. Amerallium were sequenced. • The A. paradoxum plastome differs markedly from other sequenced onion species. • A. paradoxum has long inversion in the SSC region and has lost all NADH-genes. • Shade-loving nature of A. paradoxum and loss of ndh genes could be interrelated. In this work the complete chloroplast DNAs of Allium paradoxum and Allium ursinum , two edible species of Allium subg. Amerallium (the first lineage), were sequenced, assembled, annotated, and compared with complete Allium plastomes of the second and third evolutionary lines from GenBank database. The A. ursinum plastome contains 90 predicted genes (81 unique) including 5 pseudogenes, while A. paradoxum has 88 predicted genes (79 unique) including 19 pseudogenes. The comparative analysis has revealed that the A. paradoxum plastome differs markedly from those of other species. Due to many deletions, the A. paradoxum plastome is the shortest of known for Allium species, being only 145,819 bp long. The most prominent distinctions are (1) a 4825 bp long local inversion that spans from the ndhE to the rpl32 gene in the small single copy region and (2) pseudogenization, or the loss of all NADH -genes. In contrast, the plastome of A. ursinum – a species from the first evolutionary line (as well as A. paradoxum) – resembles the Allium species of the second and third evolutionary lines, showing no large rearrangements or discrepancies in gene content. It is unclear yet whether only A. paradoxum was affected by some evolutionary events or its close relatives from both sect. Briseis and other sections of Amerallium were altered as well. We speculate the sunlight-intolerant, shade-loving nature of A. paradoxum and the impairment of the ndh genes in its plastome could be interrelated phenomena. [ABSTRACT FROM AUTHOR]