The horse pseudoautosomal region (PAR): characterization and comparison with the human, chimp and mouse PARs

The pseudoautosomal region (PAR) is a genomic segment on mammalian sex chromosomes where sequence homology mimics that seen between autosomal homologues. The region is essential for pairing and proper segregation of sex chromosomes during male meiosis. As yet, only human/chimp and mouse PARs have been characterized. The two groups of species differ dramatically in gene content and size of the PAR and therefore do not provide clues about the likely evolution and constitution of PAR among mammals. Here we characterize the equine PAR by i) isolating and arranging 71 BACs containing 129 markers (110 STS and 19 genes) into two contigs spanning the region, ii) precisely localizing the pseudoautosomal boundary (PAB), and iii) describing part of the contiguous X- and Y-specific regions. We also report the discovery of an approximately 200 kb region in the middle of the PAR that is present in the male-specific region of the Y (MSY) as well. Such duplication is a novel observation in mammals. Further, comparison of the equine PAR with the human counterpart shows that despite containing orthologs from an additional 1 Mb region beyond the human PAR1, the equine PAR is around 0.9 Mb smaller than the size of the human PAR. We theorize that the PAR varies in size and gene content across evolutionarily closely as well as distantly related mammals. Although striking differences like those observed between human and mouse may be rare, variations similar to those seen between horse and human may be prevalent among mammals.     

Rapid evolution of human pseudoautosomal genes and their mouse homologs

Comparative studies of genes in the pseudoautosomal region (PAR) of human and mouse sex chromosomes have thus far been very limited. The only comparisons that can presently be made indicate that the PARs of humans and mice are not identical in terms of gene content. Here we describe additional comparative studies of human pseudoautosomal genes and their mouse homologs. Using a somatic cell hybrid mapping panel, we have assigned the mouse homolog of the human pseudoautosomal interleukin 3 receptor alpha subunit (IL3RA) gene to mouse Chromosome (Chr) 14. Attempts to clone the mouse homolog of the human pseudoautosomal adenine nucleotide translocase-3 (ANT3) gene resulted in the isolation of the murine homologs of the human ANT1 and ANT2 genes. The mouse Ant1 and Ant2 genes are very similar in sequence to their human homologs, and we have mapped them to mouse Chromosomes (Chrs) (8 and X respectively) that exhibit conserved synteny with the chromosomes on which the human genes are located. In contrast, the homolog of ANT3 appears to be either very divergent or absent from the mouse genome. Southern blot analysis of DNA from a variety of mammalian species shows restricted conservation of human pseudoautosomal genes, a trend that also applies to the two cloned mouse homologs of these genes and to neighboring human genes in distal Xp22.3. Our observations combined with those of other workers lead us to propose a model for the evolution of the PAR that includes both rapid sequence evolution and the incremental reduction in size of the region during mammalian evolution. Received: 4 May 1995 / Accepted: 21 August 1995     

Elevated DNA sequence diversity in the genomic region of the phosphatase PPP2R3L gene in the human pseudoautosomal region

The evolution, inheritance and recombination rate of genes located in the pseudoautosomal region 1 (PAR1) is exceptional within the human genome. Pseudoautosomal genes are identical on X and Y chromosomes and are not inherited in a sex linked manner. Due to an obligatory recombination event in male meiosis, pseudoautosomal genes are exchanged frequently between X and Y chromosomes. During the isolation, characterization and sequencing of a novel gene PPP2R3L, which was classified by sequence homology as a novel member of the protein phosphatase regulatory subunit families, it became apparent that cosmids of different origin harboring this gene are highly polymorphic between individuals, both at the nucleotide level and in the number.     

Cloning and characterization of a novel mouse Siglec, mSiglec-F: differential evolution of the mouse and human (CD33) Siglec-3-related gene clusters

A novel mouse Siglec (mSiglec-F) belonging to the subfamily of Siglec-3-related Siglecs has been cloned and characterized. Unlike most human Siglec-3 (hSiglec-3)-related Siglecs with promiscuous linkage specificity, mSiglec-F shows a strong preference for alpha2-3-linked sialic acids. It is predominantly expressed in immature cells of the myelomonocytic lineage and in a subset of CD11b (Mac-1)-positive cells in some tissues. As with previously cloned Siglec-3-related mSiglecs, the lack of strong sequence similarity to a singular hSiglec made identification of the human ortholog difficult. We therefore conducted a comprehensive comparison of Siglecs between the human and mouse genomes. The mouse genome contains eight Siglec genes, whereas the human genome contains 11 Siglec genes and a Siglec-like gene. Although a one-to-one orthologous correspondence between human and mouse Siglecs 1, 2, and 4 is confirmed, the Siglec-3-related Siglecs showed marked differences between human and mouse. We found only four Siglec genes and two pseudogenes in the mouse chromosome 7 region syntenic to the Siglec-3-related gene cluster on human chromosome 19, which, in contrast, contains seven Siglec genes, a Siglec-like gene, and thirteen pseudogenes. Although analysis of gene maps and exon structures allows tentative assignments of mouse-human Siglec ortholog pairs, the possibility of unequal genetic recombination makes the assignments inconclusive. We therefore support a temporary lettered nomenclature for additional mouse Siglecs. Current information suggests that mSiglec-F is likely a hSiglec-5 ortholog. The previously reported mSiglec-3/CD33 and mSiglec-E/MIS are likely orthologs of hSiglec-3 and hSiglec-9, respectively. The other Siglec-3-like gene in the cluster (mSiglec-G) is probably a hSiglec-10 ortholog. Another mouse gene (mSiglec-H), without an apparent human ortholog, lies outside of the cluster. Thus, although some duplications of Siglec-3-related genes predated separation of the primate and rodent lineages (about 80-100 million years ago), this gene cluster underwent extensive duplications in the primate lineage thereafter.     

The Human Pseudoautosomal Region (PAR): Origin, Function and Future

The pseudoautosomal regions (PAR1 and PAR2) of the human X and Y chromosomes pair and recombine during meiosis. Thus genes in this region are not inherited in a strictly sex-linked fashion. PAR1 is located at the terminal region of the short arms and PAR2 at the tips of the long arms of these chromosomes. To date, 24 genes have been assigned to the PAR1 region. Half of these have a known function. In contrast, so far only 4 genes have been discovered in the PAR2 region. Deletion of the PAR1 region results in failure of pairing and male sterility. The gene SHOX (short stature homeobox-containing) resides in PAR1. SHOX haploinsufficiency contributes to certain features in Turner syndrome as well as the characteristics of Leri-Weill dyschondrosteosis. Only two of the human PAR1 genes have mouse homologues. These do not, however, reside in the mouse PAR1 region but are autosomal. The PAR regions seem to be relics of differential additions, losses, rearrangements and degradation of the X and Y chromosome in different mammalian lineages. Marsupials have three homologues of human PAR1 genes in their autosomes, although, in contrast to mouse, do not have a PAR region at all. The disappearance of PAR from other species seems likely and this region will only be rescued by the addition of genes to both X and Y, as has occurred already in lemmings. The present review summarizes the current understanding of the evolution of PAR and provides up-to-date information about individual genes residing in this region.     

Computational searches for missing orthologs: the case of S100A12 in mice

The interaction of the Ca2+-binding protein S100A12 with RAGE (receptor of advanced glycation endproducts) has been considered as a novel proinflammatory axis, since blockage of RAGE/S100A12 ligation suppresses chronic cellular activation and tissue injury in mouse models. However, the existence of a murine S100A12 ortholog is unknown. Because experimental approaches failed to identify it, we started an analysis of gene locus evolution. Human S100A12 is localized in the S100 gene cluster between S100A8 and S100A9, which are neighbors in both mouse and human. Confirming identical gene order, we found a DNA region between the murine S100A8 and S100A9 genes that is 60.9% identical to a region of the human S100A12 gene, including the first exon. Instead of the second and third exon, we found homology to a region close to the human S100A9 locus. To exclude a murine S100A12 ortholog elsewhere in the genome, we used human S100A12 as query for TBlastN homology searches. The matches were either too short, or identity was too low, or they could clearly be identified as distinct S100 genes. Obviously, an S100A12 ortholog is neither present in mouse nor rat, indicating that S100A12 has been lost during rodent evolution, probably due to a deletion.     

Significant positive correlation between the recombination rate and GC content in the human pseudoautosomal region.

This paper establishes that recombination drives the evolution of GC content in a significant way. Because the human P-arm pseudoautosomal region (PAR1) has been shown to have a high recombination rate, at least 20-fold more frequent than the genomic average of approximately 1 cM/Mb, this region provides an ideal system to study the role of recombination in the evolution of base composition. Nine non-coding regions of PAR1 are analyzed in this study. We have observed a highly significant positive correlation between the recombination rate and GC content (rho = 0.837, p < or = 0.005). Five regions that lie in the distal part of PAR1 are shown to be significantly higher than genomic average divergence. By comparing the intra- and inter-specific AT->GC -GC->AT ratios, we have detected no fixation bias toward GC alleles except for L254915, which has excessive AT-->GC changes in the human lineage. Thus, we conclude that the high GC content of the PAR1 genes better fits the biased gene conversion (BGC) model.     

Isolation and characterization of the mouse ubiquitin-specific protease Usp15

We have characterized the mouse ortholog of the human ubiquitin-specific protease USP15. Mouse Usp15 consists of 981 amino acids with a predicted molecular mass of 112 kDa, contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes, and is 98% identical/99% similar to human USP15. Usp15 shares 59.5% identity/75.5% sequence similarity with the mouse Unp(Usp4) oncoprotein. Recombinant Usp15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to glutathione S-transferase. Usp15 can also cleave the ubiquitin-proline bond, as can USP15 and Usp4. Alignment of mouse and human Usp15 and Usp4 protein sequences suggested that Usp15/USP15 may be alternately spliced in a manner analogous to Usp4. Sequence analysis of RT-PCR products from several human and mouse cell lines and tissues revealed alternate splicing in all cells studied. Northern blot analysis of both mouse and human Usp15 revealed two differently sized mRNAs in all tissues examined, owing to alternate polyadenylation sites spaced by 1.5 kb. Chromosomal mapping by interspecific backcross analysis localized the Usp15 gene to the distal region of mouse Chromosome (Chr) 10. This region is syntenic with human Chr 12q24, the location of human USP15, and a different location to Unp(Usp4) (Chr 9). Identification of the mouse Usp15 gene (>69.5 kb) and human USP15 gene (145 kb) sequences in genome databases reveals that both are composed of 22 exons with identical splice sites, and both have an exon/intron structure identical to the mouse Usp4 gene, including the alternately spliced exon. Phylogenetic studies suggest that a sequence currently identified as a chicken Usp4 ortholog is in fact a USP15 ortholog, while bona-fide chicken, cow, and rat Usp4 orthologs can be identified in EST databases.     

Genomic organization of the interleukin-1 locus

Recent additions have expanded the interleukin (IL)-1 gene family to 10 members. We have determined the order, orientation, and intergenic distance of the nine IL-1 family genes that lie on human chromosome 2. We report cDNA sequences for the mouse orthologs of three of these genes. The order and orientation of the mouse genes have been mapped, and the mouse locus compared with the human locus. There is a break in the mouse locus of > 100 kb, compared with the human locus, located between Il1b and the most centromere-proximal of the novel mouse genes. The mouse seems to be missing an ortholog of human IL1F7.     

Cloning and expression analysis of a novel gene, RP42, mapping to an autism susceptibility locus on 6q16

We isolated a novel mouse gene, RP42, in a systematic search for genes expressed in proliferating neuroblasts whose human orthologs map to susceptibility loci for autism. This gene is intronless and encodes a putative 259-amino-acid protein that exhibits 30-36% overall sequence identity to a fission yeast and a nematode protein (GenPept Accession Nos. CAA17006 and CAB54261). Nevertheless, no homology to any known gene was found. RP42 has developmentally regulated expression, particularly in proliferating neuroblasts from which neocortical neurons originate. Its human ortholog is located in a cluster of embryonic neuronally expressed genes on the 6q16 chromosome, making it a positional candidate susceptibility gene for autism.     

The pseudoautosomal regions, SHOX and disease

The pseudoautosomal regions represent blocks of sequence identity between the mammalian sex chromosomes. In humans, they reside at the ends of the X and Y chromosomes and encompass roughly 2.7 Mb (PAR1) and 0.33 Mb (PAR2). As a major asset of recently available sequence data, our view of their structural characteristics could be refined considerably. While PAR2 resembles the overall sequence composition of the X chromosome and exhibits only slightly elevated recombination rates, PAR1 is characterized by a significantly higher GC content and a completely different repeat structure. In addition, it exhibits one of the highest recombination frequencies throughout the entire human genome and, probably as a consequence of its structural features, displays a significantly faster rate of evolution. It therefore represents an exceptional model to explore the correlation between meiotic recombination and evolutionary forces such as gene mutation and conversion. At least twenty-nine genes lie within the human pseudoautosomal regions, and these genes exhibit 'autosomal' rather than sex-specific inheritance. All genes within PAR1 escape X inactivation and are therefore candidates for the etiology of haploinsufficiency disorders including Turner syndrome (45,X). However, the only known disease gene within the pseudoautosomal regions is the SHORT STATURE HOMEBOX (SHOX) gene, functional loss of which is causally related to various short stature conditions and disturbed bone development. Recent analyses have furthermore revealed that the phosphorylation-sensitive function of SHOX is directly involved in chondrocyte differentiation and maturation.     

Recombination in the Human Pseudoautosomal Region PAR1

The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.     

The Ortholog Conjecture Is Untestable by the Current Gene Ontology but Is Supported by RNA Sequencing Data

The ortholog conjecture posits that orthologous genes are functionally more similar than paralogous genes. This conjecture is a cornerstone of phylogenomics and is used daily by both computational and experimental biologists in predicting, interpreting, and understanding gene functions. A recent study, however, challenged the ortholog conjecture on the basis of experimentally derived Gene Ontology (GO) annotations and microarray gene expression data in human and mouse. It instead proposed that the functional similarity of homologous genes is primarily determined by the cellular context in which the genes act, explaining why a greater functional similarity of (within-species) paralogs than (between-species) orthologs was observed. Here we show that GO-based functional similarity between human and mouse orthologs, relative to that between paralogs, has been increasing in the last five years. Further, compared with paralogs, orthologs are less likely to be included in the same study, causing an underestimation in their functional similarity. A close examination of functional studies of homologs with identical protein sequences reveals experimental biases, annotation errors, and homology-based functional inferences that are labeled in GO as experimental. These problems and the temporary nature of the GO-based finding make the current GO inappropriate for testing the ortholog conjecture. RNA sequencing (RNA-Seq) is known to be superior to microarray for comparing the expressions of different genes or in different species. Our analysis of a large RNA-Seq dataset of multiple tissues from eight mammals and the chicken shows that the expression similarity between orthologs is significantly higher than that between within-species paralogs, supporting the ortholog conjecture and refuting the cellular context hypothesis for gene expression. We conclude that the ortholog conjecture remains largely valid to the extent that it has been tested, but further scrutiny using more and better functional data is needed.     

MSOAR: a high-throughput ortholog assignment system based on genome rearrangement.

The assignment of orthologous genes between a pair of genomes is a fundamental and challenging problem in comparative genomics, since many computational methods for solving various biological problems critically rely on bona fide orthologs as input. While it is usually done using sequence similarity search, we recently proposed a new combinatorial approach that combines sequence similarity and genome rearrangement. This paper continues the development of the approach and unites genome rearrangement events and (post-speciation) duplication events in a single framework under the parsimony principle. In this framework, orthologous genes are assumed to correspond to each other in the most parsimonious evolutionary scenario involving both genome rearrangement and (post-speciation) gene duplication. Besides several original algorithmic contributions, the enhanced method allows for the detection of inparalogs. Following this approach, we have implemented a high-throughput system for ortholog assignment on a genome scale, called MSOAR, and applied it to human and mouse genomes. As the result will show, MSOAR is able to find 99 more true orthologs than the INPARANOID program did. In comparison to the iterated exemplar algorithm on simulated data, MSOAR performed favorably in terms of assignment accuracy. We also validated our predicted main ortholog pairs between human and mouse using public ortholog assignment datasets, synteny information, and gene function classification. These test results indicate that our approach is very promising for genome-wide ortholog assignment. Supplemental material and MSOAR program are available at http://msoar.cs.ucr.edu.     

Early diversification of the TNF superfamily in teleosts: genomic characterization and expression analysis

The TNF superfamily (TNFSF) of proteins are cytokines involved in diverse immunological and developmental pathways. Little is known about their evolution or expression in lower vertebrate species. Bioinformatic searches of Zebrafish, Tetraodon, and Fugu genome and other teleost expressed sequence tag databases identified 44 novel gene sequences containing a TNF homology domain. This work reveals the following: 1) teleosts possess orthologs of BAFF, APRIL, EDA, TWEAK, 4-1BBL, Fas ligand, LIGHT, CD40L, RANKL, and possibly TL1A; 2) the BAFF-APRIL subfamily is enriched by a third member, BALM, unique to fish; 3) orthologs of lymphotoxins alpha and beta were not clearly identified in teleosts and are substituted by a related ligand, TNF-New; 4) as many as four TRAIL-like genes are present in teleosts, as compared with only one in mammals; and 5) T cell activation ligands OX40L, CD27L, CD30L, and GITRL were not identified in any fish species. Finally, we characterize mRNA expression of TNFSF members CD40L, LIGHT, BALM, APRIL, Fas ligand, RANKL, TRAIL-like, and TNF-New in rainbow trout, Oncorhynchus mykiss, immune and nonimmune tissues. In conclusion, we identified a total of 14 distinct TNFSF members in fishes, indicating expansion of this superfamily before the divergence of bony fish and tetrapods, approximately 360-450 million years ago. Based on these findings, we extend a model of TNFSF evolution and the co-emergence of the vertebrate adaptive immune system.     

Evolutionary rate of a gene affected by chromosomal position.

Genes evolve at different rates depending on the strength of selective pressure to maintain their function. Chromosomal position can also have an influence [1] [2]. The pseudoautosomal region (PAR) of mammalian sex chromosomes is a small region of sequence identity that is the site of an obligatory pairing and recombination event between the X and Y chromosomes during male meiosis [3] [4] [5] [6]. During female meiosis, X chromosomes can pair and recombine along their entire length. Recombination in the PAR is therefore approximately 10 times greater in male meiosis compared with female meiosis [4] [5] [6]. The gene Fxy (also known as MID1 [7]) spans the pseudoautosomal boundary (PAB) in the laboratory mouse (Mus musculus domesticus, C57BL/6) such that the 5' three exons of the gene are located on the X chromosome but the seven exons encoding the carboxy-terminal two-thirds of the protein are located within the PAR and are therefore present on both the X and Y chromosomes [8]. In humans [7] [9], the rat, and the wild mouse species Mus spretus, the gene is entirely X-unique. Here, we report that the rate of sequence divergence of the 3' end of the Fxy gene is much higher (estimated at 170-fold higher for synonymous sites) when pseudoautosomal (present on both the X and Y chromosomes) than when X-unique. Thus, chromosomal position can directly affect the rate of evolution of a gene. This finding also provides support for the suggestion that regions of the genome with a high recombination frequency, such as the PAR, may have an intrinsically elevated rate of sequence divergence.