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Pancreatic cancer, the most lethal malignant tumor, is notorious for its poor prognosis and metastatic potential. Non-coding RNAs (ncRNAs) are reported to play key roles in cancer metastasis. In this study, miRNA and gene expression profiles between metastatic pancreatic cancer cell M8 and its parental cell BxPC.3 were determined. Using differential expression analysis, survival analysis, target gene prediction, pathway enrichment analysis, intersection analysis and correlation analysis, hsa-miR-30d-5p/GJA1 axis was identified as the most potential pathway involved in metastasis of pancreatic cancer. Subsequently, two upstream lncRNAs (HELLPAR and OIP-AS1) and four upstream pseudogenes (AC093616.1, AC009951.1, TMEM183B and PABPC1P4) of hsa-miR-30d-5p/GJA1 axis were predicted and were then identified via assessment of RNA-RNA expression relationship. Furthermore, CTNNA1, CTNNB1 and CTNND1 were regarded as three crucial molecules to be participated in hsa-miR-30d-5p/GJA1-mediated metastatic potential in pancreatic cancer. In conclusion, we established a novel lncRNA/pseudogene-hsa-miR-30d-5p-GJA1 regulatory network linked to metastasis of pancreatic cancer.

The protein-coding genes and pseudogenes of Cuscuta australis had the diverse contribution to the formation and evolution of parasitism. The codon usage pattern analysis of these two type genes could be used to understand the gene transcription and translation. In this study, we systematically analyzed the codon usage patterns of protein-coding sequences and pseudogenes sequences in C. australis. The results showed that the high frequency codons of protein coding sequences and pseudogenes had the same A/U bias in the third position. However, these two sequences had converse bias at the third base in optimal codons: the protein coding sequences preferred G/C-ending codons while pseudogene sequences preferred A/U-ending codons. Neutrality plot and effective number of codons plot revealed that natural selection played a more important role than mutation pressure in two sequences codon usage bias. Furthermore, the gene expression level had a significant positive correlation with codon usage bias in C. australis. Highly-expressed protein coding genes exhibited a higher codon bias than lowly-expressed genes. Meanwhile, the high-expression genes tended to use G/C-ending synonymous codons. This result further verified the optimal codons usage bias and its correlation with the gene expression in C. australis.

Described as "junk" DNA, pseudogenes are dead structures of previously active genes present in genomes. Pseudogenes are categorized into two main classes: processed pseudogenes, formed through retrotransposition, and non-processed pseudogenes, typically originated from gene decay following duplication events. The term "processed pseudogene" has changed to "retrocopy" since they are likely to evolve new functional roles and became a retrogene. Here, we surveyed 38,080 retrocopies from chimpanzee, dog, human, mouse, and rat genomes to assess their potential adaptive value. The retrocopies inserted in the same chromosome of the parental gene have higher chances of remain potentially "active" (absence of premature stop codons and frameshifts) (~26.1%), while those placed into a different chromosome have a twofold decrease chance of continuing potentially "active" (~7.52%). The genomic context of their placement seems associated with their expression. Retrocopies placed in intragenic regions and the same sense of the "host" gene have higher chances of being expressed relative to other genomic contexts. The proximity of retrocopies to their parental gene is associated with a lower decay rate, and their location likely influence their expression. Thus, despite their unclear role, retrocopies are probably involved in adaptive processes. Our results evidence natural selection acting in retrocopies.

CACX is one of the most common cancer affecting women world-wide. Here, expression microarray analysis revealed 8 over-expressed transcribed pseudogenes ( GBP1P1 , HLA-DRB6 , HLA H , SLC6A10P , NAPSB, KRT16P2 , PTTG3P and RNF126P1), down-regulated 7 lincRNAs ( H19 , MIR100HG , MEG3 , DIO3OS , HOXA11-AS , CD27-AS1 and EPB41L4A-AS ) and 6 snoRNAs ( SNORD97 , SNORD3A , SNORD3C , SNORD3D , SNORA12 and SCARNA9 ) as DEncGs (log2 fold-change ≥ ±1.0) in CACX. Consequently, down-regulation of lincRNA MEG3 and over-expression of pseudogenes, GBP1P1 and PTTG3P in the microarray analysis were found concordant with the real-time quantitative PCR results upon validation. Then, Ingenuity® Pathway analysis (IPA®) analysis with deregulated DEncGs identified functionally important gene, H19 . Further, validation ( n = 52) of expression confirmed frequent downregulation of H19 with significant association with its deletion (LOH) and promoter methylation ( n = 128) in CACX. Moreover, clinico-pathological analysis found Indian CACX patients ( n = 26) with alterations of H19 by deletion or, promoter methylation with concomitant low expression have poor prognosis.

Estrogen drives key transcriptional changes in breast cancer and stimulates breast cancer cells' growth with multiple mechanisms to coordinate transcription and translation. In addition to protein-coding transcripts, estrogen can regulate long non-coding RNA (lncRNA) transcripts, plus diverse non-coding RNAs including antisense, enhancer, and intergenic. LncRNA genes comprise the majority of human genes. The accidental, or regulated, translation of their short open reading frames by ribosomes remains a controversial topic. Here we report for the first time an integrated analysis of RNA abundance and ribosome occupancy level, using Ribo-seq combined with RNA-Seq, in the estrogen-responsive, estrogen receptor α positive, human breast cancer cell model MCF7, before and after hormone treatment. Translational profiling can determine, in an unbiased manner, which fraction of the genome is actually translated into proteins, as well as resolving whether transcription and translation respond concurrently, or differentially, to estrogen treatment. Our data showed specific transcripts more robustly detected in RNA-Seq than in the ribosome-profiling data, and vice versa, suggesting distinct gene-specific estrogen responses at the transcriptional and the translational level, respectively. Here, we showed that estrogen stimulation affects the expression levels of numerous lncRNAs, but not their association with ribosomes, and that most lncRNAs are not ribosome-bound. For the first time, we also demonstrated the transcriptional and translational response of expressed pseudogenes to estrogen, pointing to new perspectives for drug-target development in breast cancer in the future.

The ancestors of Cetacea underwent profound morpho-physiological alterations. By displaying an exclusive aquatic existence, cetaceans evolved unique patterns of locomotor activity, vigilant behaviour, thermoregulation and circadian rhythmicity. Deciphering the molecular landscape governing many of these adaptations is key to understand the evolution of phenotypes. Here, we investigate Cortistatin (CORT), a neuropeptide displaying an important role in mammalian biorhythm regulation. This neuropeptide is a known neuroendocrine factor, stimulating slow-wave sleep, but also involved in the regulation of energy metabolism and hypomotility inducement. We assessed the functional status of CORT in 359 mammalian genomes (25 orders), including 30 species of Cetacea. Our findings indicate that cetaceans and other mammals with atypical biorhythms, thermal constraints and/or energy metabolism, have accumulated deleterious mutations in CORT. In light of the pleiotropic action of this neuropeptide, we suggest that this inactivation contributed to a plethora of phenotypic adjustments to accommodate adaptive solutions to specific ecological niches.

Here we report a comprehensive analysis of the vomeronasal receptor repertoire in pigs. We identified a total of 25 V1R sequences consisting of 10 functional genes, 3 pseudogenes, and 12 partial genes, while functional V2R and FPR genes were not present in the pig genome. Pig V1Rs were classified into three subfamilies, D, F, and J. Using direct high resolution sequencing-based typing of all functional V1Rs from 10 individuals of 5 different breeds, a total of 24 SNPs were identified, indicating that the allelic diversity of V1Rs is much lower than that of the olfactory receptors. A high expression level of V1Rs was detected in the vomeronasal organ (VNO) and testes, while a low expression level of V1Rs was observed in all other tissues examined. Our results showed that pigs could serve as an interesting large animal model system to study pheromone-related neurobiology because of their genetic simplicity.

Actinobacillus spp. are Gram-negative bacteria associated with mucosal membranes. While some are commensals, others can cause important human and animal diseases. A. pleuropneumoniae causes severe fibrinous hemorrhagic pneumonia in swine but not systemic disease whereas other species invade resulting in septicemia and death. To understand the invasive phenotype of Actinobacillus spp., complete genomes of eight isolates were obtained and pseudogenomes of five isolates were assembled and annotated. Phylogenetically, A. suis isolates clustered by surface antigen type and were more closely related to the invasive A. ureae, A. equuli equuli, and A. capsulatus than to the other swine pathogen, A. pleuropneumoniae. Using the LS-BSR pipeline, 251 putative virulence genes associated with serum resistance and invasion were detected. To our knowledge, this is the first genome-wide study of the genus Actinobacillus and should contribute to a better understanding of host tropism and mechanisms of invasion of pathogenic Actinobacillus and related genera.

C/D box small nucleolar RNAs (snoRNAs) guide site-specific 2′-O-methylation of RNAs. Nearly all C/D box snoRNAs with known targets are involved in rRNA modification. In vertebrates, snoRNAs are encoded in introns of various genes and their processing is coupled with splicing of host gene pre-mRNA. Here, the genes encoding C/D box snoRNAs that guide 2′-O-methylation of rRNA were identified and analyzed in vertebrate genomes. The number of copies of most C/D box snoRNA genes proved to be lower in placental mammals compared to other vertebrates. This can be due to smaller oocytes and accordingly lower number of ribosomes in them in eutherians. The targets of snoRNAs encoded by single-copy and multiple-copy genes proved to have different distribution in rRNAs. The causes of this difference are discussed. In some cases, the transcripts of homologous C/D box RNA genes were shown to guide the modification of neighboring nucleotides in rRNA. C/D box snoRNA pseudogenes were found in all vertebrate classes. Three novel C/D box snoRNAs were found in Xenopus tropicalis that may guide 2′-O-methylation of Xenopus-specific rRNA sites. A list of 922 annotated C/D box snoRNA genes is presented.

Demonstrating an association between a polymorphism and a disease phenotype through case-control studies requires reliable large-scale genotyping, but accurate measurement of copy number variation has proven to be technically challenging. Here we build on our previous experience with Paralogue Ratio Tests (PRT) to develop PRT copy number determination at the CCL3L1/CCL4L1 copy number variant. A multiplex PRT assay based on four independent comparative PCRs results in a convenient, accurate and robust method of multiallelic copy number measurement suitable for use in large-scale case-control studies, which can unambiguously assign virtually all samples tested to discrete copy number classes.

We applied a comprehensive data mining strategy to examine the repertoires of rat and mouse odorant receptors (ORs) and type 1 pheromone receptors (V1Rs) using the mm5 and rn3 genome respectively. 1576 rat OR genes were identified, including 292 pseudogenes. The rat V1R repertoire is composed of 115 intact genes and 72 pseudogenes. The mouse OR and V1R database were updated using the new assembly mm5, from which 1375 mouse ORs and 308 V1Rs were identified, with more than a hundred putative pseudogenes from mm2 now identified as intact because of the higher sequence quality. With this new data we have conducted a series of genomic analyses of the OR and V1R genes from mouse and rat. Orthologous OR clusters were identified in mouse and rat and comparison analysis was performed at three incremental levels: families, coding sequences, and motifs. At the family level, we found that V1R genes have more species-specific families than OR genes. About 20 percent of intact V1R genes have no orthologous counterpart in the same family, whereas less than 1 percent of intact ORs are similarly isolated. At the coding sequence level, OR genes are more conserved between mouse and rat than V1R genes. OR genes share greater similarity with their orthologous counterparts than with their closest neighbor, whereas V1R genes show the opposite tendency. Motifs were identified to obtain biological insights. Motifs specific for species or families were found in OR and V1R genes, which may result in the differential pheromone-dependent behaviors and perception of odors between mouse and rat.

We have discovered a family of small secreted proteins in Homo sapiens and Mus musculus. The IGF-like (IGFL) genes encode proteins of approximately 100 amino acids that contain 11 conserved cysteine residues at fixed positions, including two CC motifs. In H. sapiens, the family is composed of four genes and two pseudogenes that are referred as IGFL1 to IGFL4 and IGFL1P1 and IGFL1P2, respectively. Human IGFL genes are clustered together on chromosome 19 within a 35-kb interval. M. musculus has a single IGFL family member that is located on chromosome 7. Further, evolutionary analysis shows a lack of direct orthology between any of the four human members and the mouse gene. This relationship between the mouse and the human family members suggests that the multiple members in the human complement have arisen from recent duplication events that appear limited to the primate lineage. Structural considerations and sequence comparisons would suggest that IGFL proteins are distantly related to the IGF superfamily of growth factors. IGFL mRNAs display specific expression patterns; they are expressed in fetal tissues, breast, and prostate, and in many cancers as well, and this pattern is consistent with that of the IGF family members.

Rhox is a recently identified cluster of 12 X-linked homeobox genes in mice. The expression pattern of Rhox genes during postnatal testis development corresponds to their chromosomal position, much like the colinear gene regulation of the Hox gene clusters during animal embryonic development. We here report the identification of 18 additional Rhox genes and 3 pseudogenes in mice. Comparative analyses of the mouse, rat, human, dog, cow, opossum, and chicken genomes suggest that the Rhox cluster originated in the common ancestor of primates and rodents. It subsequently underwent two remarkable expansions, first in the common ancestor of mice and rats and then in mice. Positive selection promoting amino acid substitutions was detected in some young Rhox genes, suggesting adaptive functional diversification. The recent expansions of the Rhox cluster provide an opportunity to study the mechanism and origin of colinear gene regulation, but they may also undermine the utility of mouse models for understanding the development and physiology of the human reproductive system.

The TSPY gene is conserved in placental mammals and encodes the testis-specific protein, Y encoded. Within the testis, TSPY expression is restricted to germ cells, and it is assumed that TSPY plays a role in the proliferation of germ cells. Since it was first discovered in humans, TSPY orthologous gene families have been subsequently characterized in many mammalian lineages. In contrast to the situation in cattle and primates, in which TSPY is organized in a moderately repetitive cluster, including functional members and pseudogenes, a peculiar situation is observed in rodents, in which Tspy has been become low or single copy and degenerated to a pseudogene in some species of the subgenus Mus. We have extended this approach and investigated Tspy gene evolution in the Syrian hamster (Mesocricetus auratus) and the Mongolian gerbil (Meriones unguiculatus). Whereas the Syrian hamster Tspy is functionally conserved, organized in multiple copies, and expressed only in testis, the closely related Mongolian gerbil possesses a single-copy pseudogene that is unable to generate a functional transcript. Thus, the Tspy locus has degenerated at least twice at different points of rodent evolution, strongly supporting the hypothesis that the decay of Y-chromosomal genes is an intrinsic evolutionary process. TSPY is the first example of a Y-chromosomal tandem repetitive gene whose decay could be studied in two independent mammalian lineages.

Most members of the large family of rhodopsin-like G-protein-coupled receptors possess an evolutionarily conserved Asp-Arg-Tyr (DRY) motif in the C-terminal region of the third transmembrane domain. Mutations of residues within this motif usually abolish receptor function and, when they occur naturally, can even cause human diseases. By analyzing over 100 mammalian orthologs of the chemoattractant receptor GPR33 we identified several polymorphic and fixed sequence variations within the DRY motif. Unexpectedly, the naturally occurring mutation of Arg(3.50) to His in mouse GPR33 showed no difference from the wild-type receptor in several functional tests. Sequence analysis of GPR33 from Asian house mice revealed the polymorphic existence of Arg(3.50) and His(3.50) alleles in wild-trapped populations, further supporting the functional equivalence of both allelic variants. In contrast, the Arg(3.50) to Gly mutation found in hamster GPR33 inactivates the receptor and may have contributed to pseudogenization of this gene in this species. Functional data with GPR33 variants indicate different receptor- and context-specific consequences of DRY mutations. Our study also reveals GPR33 as a new example illustrating missense mutations as a first step in the pseudogenization process.

The sperm mitochondrial cysteine-rich protein (SMCP) is a rapidly evolving cysteine- and proline-rich protein that is localized in the mitochondrial capsule and enhances sperm motility. The sequences of the SMCP protein, gene, and mRNA in a variety of mammals have been compared to understand their evolution and regulation. SMCP can now be reliably identified by its tripartite structure including a short amino-terminal segment; a central segment containing short tandem repeats rich in cysteine, proline, glutamine, and lysine; and a C-terminal segment containing no repeats, few cysteines, and a C-terminal lysine. The SMCP gene is located in the epidermal differentiation complex (EDC), a large gene cluster that functions in forming epithelial barriers. Similarities in chromosomal location, molecular function, intron–exon structure, and protein organization argue that SMCP originated from an EDC gene and acquired spermatogenic cell-specific transcriptional and translational regulation and a novel cellular function in sperm motility. The SMCP 5′ UTR and 3′ UTR contain conserved elements and uORFs that may function in cytoplasmic regulation of gene expression, and the levels of SMCP mRNA in human are much lower than in other mammals, a feature of male-biased expression. The evolution of SMCP has been accompanied by changes in the sequence, number, and length of repeat units, including three alleles in dogs. The major proteins associated with the mitochondrial capsule, SMCP and phospholipid hydroperoxide glutathione peroxidase, provide outstanding examples of changes in cellular function driven by selective pressures on sperm motility, an important determinant of male reproductive success.

Proteolytic enzymes are implicated in multiple physiological and pathological processes. The availability of the sequence of the chimpanzee genome has allowed us to determine that the chimpanzee degradome—the repertoire of protease genes from this organism—is composed of at least 559 protease and protease-like genes and is virtually identical to that of human, containing 561 genes. Despite the high degree of conservation between both genomes, we have identified important differences that vary from deletion of whole genes to small insertion/deletion events or single nucleotide changes that lead to the specific gene inactivation in one species, mostly affecting immune system genes. For example, the genes encoding PRSS33/EOS, a macrophage serine protease conserved in most mammals, and GGTLA1 are absent in chimpanzee, while the gene for metalloprotease MMP23A, located in chromosome 1p36, has been specifically duplicated in the human genome together with its neighbor gene CDC2L1. Other differences arise from single nucleotide changes in protease genes, such as NAPSB and CASP12, resulting in the presence of functional genes in chimpanzee and pseudogenes in human. Finally, we have confirmed that the Trypanosoma lytic factor HPR is inactive in chimpanzee, likely contributing to the susceptibility of chimpanzees to T. brucei infection. This study provides the first analysis of the chimpanzee degradome and might contribute to the understanding of the molecular bases underlying variations in host defense mechanisms between human and chimpanzee.

Ficolin is a collagenous lectin which plays a crucial role in innate immunity. Three and two ficolins have been identified in human and mice, respectively. To identify the mouse homologue of human H-ficolin and to elucidate the orthology between mouse ficolins A/B and human L-/M-ficolins, the gene structures were explored. The mouse homologue of the H-ficolin gene was identified as a pseudogene on chromosome 4. The mouse ficolin A gene was located far from the ficolin B gene on chromosome 2, whereas the human L-ficolin and M-ficolin genes were close in the region homologous to the ficolin B locus. Together with the exon-intron structures and the phylogenetic tree, these results suggest that ficolin B is the mouse orthologue of M-ficolin and that the genes encoding serum-type ficolins, ficolin A and L-ficolin, were generated independently from the ficolin B/M-ficolin lineage each in mice and primates.

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.

Nanog is a recently discovered ANTP class homeobox gene. Mouse Nanog is expressed in the inner cell mass and in embryonic stem cells and has roles in self-renewal and maintenance of pluripotency. Here we describe the location, genomic organization, and relative ages of all human NANOG pseudogenes, comprising ten processed pseudogenes and one tandem duplicate. These are compared to the original, intact human NANOG gene. Eleven is an unusually high number of pseudogenes for a homeobox gene and must reflect expression in the human germ line. A pseudogene orthologous to NANOGP4 was found in chimpanzee and an expressed pseudogene in macaque. Examining pseudogenes of differing ages gives insight into pseudogene decay, which involves an excess of deletion mutations over insertions. The mouse genome has two processed pseudogenes, which are not clear orthologues of the primate pseudogenes.

The cytotoxic activity of mouse natural killer cells is regulated in part through cell surface molecules belonging to the Ly49 multigene family. In mice, the genomic sequence of the Ly49 gene cluster has been examined in detail and this analysis provided a model of the expansion of this multigene family. In the present study, we have analyzed a 1.8-Mb region of the draft rat genome revealing surprising differences in size and gene content between the mouse and the rat Ly49 clusters. The rat cluster contains at least 36 Ly49 genes, including pseudogenes, while dot-plot analysis of the cluster reveals an equidistant spacing of genes, suggesting that duplication of genes in the cluster occurred through a mechanism similar to that in the mouse. Phylogenetic analysis of the predicted rat genes reveals a number of distinct gene clusters and indicates that the majority of gene duplication events occurred after the divergence of mice and rats. Thus, the rodent Ly49 locus is subject to extremely rapid gene amplification and diversification.

Cytochrome b5/cytochrome b5 reductase (cb5/cb5r) is a cytosolic fusion protein between the hemoprotein cytochrome b5 and the flavoprotein cytochrome b5 reductase. We describe the identification and characterization of a novel splice variant of cb5/cb5r in the mouse and rat and show that expression of the variant is conserved in both species but is not expressed in human tissue. Characterization of the exon structure of cb5/cb5r indicated that the variant was due to the deletion of the whole of exon 12, thus the variant was named cb5/cb5rdelta12. Exon 12 codes for the flavin-adenine dinucleotide binding domain of cb5/cb5r. Expression analysis revealed the transcript of cb5/cb5rdelta12 in mouse and rat testis, brain, and skeletal muscle and also in the male germ line. We postulate that cb5/cb5rdelta12 may function in a dominant negative fashion, limiting the amount of damage caused by the production of reactive oxygen species by cb5/cb5r.

We identified 971 olfactory receptor (OR) genes in the dog genome, estimated to constitute approximately 80% of the canine OR repertoire. This was achieved by directed genomic DNA cloning of olfactory sequence tags as well as by mining the Celera canine genome sequences. The dog OR subgenome is estimated to have 12% pseudogenes, suggesting a functional repertoire similar to that of mouse and considerably larger than for humans. No novel OR families were discovered, but as many as 34 gene subfamilies were unique to the dog. "Fish-like" Class I ancient ORs constituted 18% of the repertoire, significantly more than in human and mouse. A set of 122 dog-human-mouse ortholog triplets was identified, with a relatively high fraction of Class I ORs. The elucidation of a large portion of the canine olfactory receptor gene superfamily, with some dog-specific attributes, may help us understand the unique chemosensory capacities of this species.

International audience; The translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF), is encoded by a gene (Tpt1) that is highly conserved throughout phylogeny. TCTP is implicated in cell growth, acute allergic response, and apoptosis. In the present study, seven putative Tpt1 genes with different chromosomal localizations were identified in the mouse genome. In six of them, analysis of the 5' and 3' untranslated regions revealed the presence of flanking direct repeats and residual poly(A) tails typical of pseudogenes. Only three of the seven genes can produce a protein of the expected molecular weight. We isolated the genomic DNA of these three genes to analyze their sequence, genomic organization, and in vitro promoter activity. We found that mouse Tpt1 is localized on chromosome 14 with a canonical intron-exon organization, a functional promoter, and only one transcript that is ubiquitously expressed in all tissues.

The human (nuclear) genome encodes at least 79 mitochondrial ribosomal proteins (MRPs), which are imported into the mitochondria. Using a comprehensive approach, we find 41 of these give rise to 120 pseudogenes in the genome. The majority of the MRP pseudogenes are of processed origin and can be aligned to match the entire coding region of the functional MRP mRNAs. One processed pseudogene was found to have originated from an alternatively spliced mRNA transcript. We also found two duplicated pseudogenes that are transcribed in the cell as confirmed by screening the human EST database. We observed a significant correlation between the number of processed pseudogenes and the gene CDS length (R = -0.40; p < 0.001), i.e., the relatively shorter genes tend to have more processed pseudogenes. There is also a weaker correlation between the number of processed pseudogenes and the gene CDS GC content. Our study provides a catalogue of human MRP pseudogenes, which will be useful in the study of functional MRP genes. It also provides a molecular record of the evolution of these genes. More details are available at http://pseudogene.org/.

Extracellular signal-regulated kinase 3 (ERK3) is a distantly related member of the mitogen-activated protein (MAP) kinase family of serine/threonine kinases. Here, we report the characterization of the genomic loci encoding ERK3 in mice and humans. The mouse ERK3 gene ( Mapk6 ) spans more than 20 kb and is split into six exons. Its structure is similar to that of the human MAPK6 gene, which extends over 40 kb. We also identified and characterized a mouse Mapk6 processed pseudogene. In humans, database analysis has revealed the presence of six MAPK6 processed pseudogenes localized on four different chromosomes. We further show that the structure of MAPK6 is closely related to that of the gene encoding the homologous protein kinase p63 MAPK ( MAPK4 ), suggesting that the two genes arose by duplication. Our analysis demonstrates that the ERK3 subfamily of MAP kinase genes is composed of two functional genes, MAPK6 and MAPK4, and several pseudogenes.

Nuclear mitochondrial pseudogenes (Numts) have been found in the genome of many eukaryote species, including humans. Using a BLAST approach, we found 1105 DNA sequences homologous to mitochondrial DNA (mtDNA) in the August 2001 Goldenpath human genome database. We assembled these sequences manually into 286 pseudogenes on the basis of single insertion events and constructed a chromosomal map of these Numts. Some pseudogenes appeared highly modified, containing inversions, deletions, duplications, and displaced sequences. In the case of four randomly selected Numts, we used PCR tests on cells lacking mtDNA to ensure that our technique was free from genome-sequencing artifacts. Furthermore, phylogenetic investigation suggested that one Numt, apparently inserted into the nuclear genome 25-30 million years ago, had been duplicated at least 10 times in various chromosomes during the course of evolution. Thus, these pseudogenes should be very useful in the study of ancient mtDNA and nuclear genome evolution.

Genes encoding novel murine cysteine peptidases of the papain family C1A and related genes were cloned and mapped to mouse chromosome 13, colocalizing with the previously assigned cathepsin J gene. We constructed a460-kb phage artificial chromosome (PAC) contig and characterized a dense cluster comprising eight C1A cysteine peptidase genes, cathepsins J, M, Q, R, -1, -2, -3, and -6; three pseudogenes of cathepsins M, -1, and -2; and four genes encoding putative cysteine peptidase inhibitors related to the proregion of C1A peptidases (trophoblast-specific proteins alpha and beta and cytotoxic T-lymphocyte-associated proteins 2alpha and -beta). Because of sequence homologies of 61.9-72.0% between cathepsin J and the other seven putative cysteine peptidases of the cluster, these peptidases are classified as "cathepsin J-like". The absence of cathepsin J-like peptidases and related genes from the human genome suggests that the cathepsin J cluster arose by partial and complete gene duplication events after the divergence of primate and rodent lineages. The expression of cathepsin J-like peptidases and related genes in the cluster is restricted to the placenta only. Clustered genes are induced at specific time points, and their expression increases toward the end of gestation. The specific expression pattern and high expression level suggest an essential role of cathepsin J-like peptidases and related genes in formation and development of the murine placenta.

We have cloned and characterized two genomic loci encoding the human type II gonadotropin-releasing hormone (GnRH) receptor and RNA-binding motif protein-8 (RBM8A). In both loci the genes overlap and are in antisense orientation to each other. The locus on chromosome 1 encompasses the type II GnRH receptor gene (GNRHR2), which is composed of three exons. We found transcripts from this gene in a wide range of tissues, but they lacked a methionine initiation codon and had a stop codon in exon 2. In the antisense orientation, this locus contains RBM8A, which consists of six exons and directs the synthesis of an RBM8A protein of 173 or 174 amino acids by alternative splicing. A second locus on chromosome 14 contains pseudogenes of RBM8A and the type II GnRH receptor and probably originated from the chromosome 1 locus by retrotransposition.

We have characterized genomic loci encoding translation elongation factor 1B(alpha) (eEF1B(alpha)) in mice and humans. Mice have a single structural locus (named Eef1b2) spanning six exons, which is ubiquitously expressed and maps close to Casp8 on mouse chromosome 1, and a processed pseudogene. Humans have a single intron-containing locus, EEF1B2, which maps to 2q33, and an intronless paralogue expressed only in brain and muscle (EEF1B3). Another locus described previously, EEF1B1, is actually a processed pseudogene on chromosome 15 corresponding to an alternative splice form of EEF1B2. Our study illustrates the value of comparative mapping in distinguishing between processed pseudogenes and intronless paralogues.

The meningioma expressed antigen-6 (MGEA6) was originally identified as an immunogenic antigen in meningioma patients. Somatic hybrid panel mapping and fluorescence in situ hybridization revealed MGEA6-related sequences on different human chromosomes. Here we carry out database analysis to investigate the complexity of the MGEA6-related sequences and demonstrate the existence of a multigene family. We localized the active gene (spanning over 83 kb) to chromosome 14q and elucidated its exon/intron structure. We identified and characterized 9 processed pseudogenes on 9 different chromosomes including chromosomes 2, 3, 6, 7, 9, 10, 12, 13, and 18. We performed phylogenetic analysis and concluded that the MGEA6 pseudogenes may result from more than one retrotransposition event; we calculated divergence times of the pseudogenes to be between 21.5 and 28.9 million years ago.

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.