Presence and phylogenetic analysis of HERV-K LTR on human X and Y chromosomes: evidence for recent proliferation

The K group of human endogenous retroviruses (HERV-K) has been suggested to have a role in disease and has recently been shown to include long terminal repeat (LTR) elements that are human specific. Here we investigated the presence of HERV-K LTRs on the human X and Y chromosomes with the use of PCR on a monochromosomal somatic cell hybrid DNA panel. We report twelve such sequences on the X chromosome and ten sequences on the Y chromosome. Phylogenetic analysis reveals that clones X2, 4, 5, 6, 7, 11, 15 from the X chromosome and clones Y4, 5, 7, 10 from the Y chromosome are closely related to the human-specific members of Medstrand and Mager's cluster 9. The sequence of clone Y7 from the Y chromosome is identical with human-specific HERV-K LTR element (AC002350) from chromosome 12q24. The findings suggest recent proliferation and transposition of HERV-K LTR elements on these chromosomes. Such events may have contributed to structural change and genetic variation in the human genome. We draw attention to evolutionarily recent changes in homologies between X and Y chromosomes as a method of further investigating such transpositions.     

Evolution of Genomic Structures on Mammalian Sex Chromosomes

Throughout mammalian evolution, recombination between the two sex chromosomes was suppressed in a stepwise manner. It is thought that the suppression of recombination led to an accumulation of deleterious mutations and frequent genomic rearrangements on the Y chromosome. In this article, we review three evolutionary aspects related to genomic rearrangements and structures, such as inverted repeats (IRs) and palindromes (PDs), on the mammalian sex chromosomes. First, we describe the stepwise manner in which recombination between the X and Y chromosomes was suppressed in placental mammals and discuss a genomic rearrangement that might have led to the formation of present pseudoautosomal boundaries (PAB). Second, we describe ectopic gene conversion between the X and Y chromosomes, and propose possible molecular causes. Third, we focus on the evolutionary mode and timing of PD formation on the X and Y chromosomes. The sequence of the chimpanzee Y chromosome was recently published by two groups. Both groups suggest that rapid evolution of genomic structure occurred on the Y chromosome. Our re-analysis of the sequences confirmed the species-specific mode of human and chimpanzee Y chromosomal evolution. Finally, we present a general outlook regarding the rapid evolution of mammalian sex chromosomes.     

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.     

Fundamentally different repetitive element composition of sex chromosomes in Rumex acetosa

Background and AimsDioecious species with well-established sex chromosomes are rare in the plant kingdom. Most sex chromosomes increase in size but no comprehensive analysis of the kind of sequences that drive this expansion has been presented. Here we analyse sex chromosome structure in common sorrel (Rumex acetosa), a dioecious plant with XY₁Y₂ sex determination, and we provide the first chromosome-specific repeatome analysis for a plant species possessing sex chromosomes.MethodsWe flow-sorted and separately sequenced sex chromosomes and autosomes in R. acetosa using the two-dimensional fluorescence in situ hybridization in suspension (FISHIS) method and Illumina sequencing. We identified and quantified individual repeats using RepeatExplorer, Tandem Repeat Finder and the Tandem Repeats Analysis Program. We employed fluorescence in situ hybridization (FISH) to analyse the chromosomal localization of satellites and transposons.Key ResultsWe identified a number of novel satellites, which have, in a fashion similar to previously known satellites, significantly expanded on the Y chromosome but not as much on the X or on autosomes. Additionally, the size increase of Y chromosomes is caused by non-long terminal repeat (LTR) and LTR retrotransposons, while only the latter contribute to the enlargement of the X chromosome. However, the X chromosome is populated by different LTR retrotransposon lineages than those on Y chromosomes.ConclusionsThe X and Y chromosomes have significantly diverged in terms of repeat composition. The lack of recombination probably contributed to the expansion of diverse satellites and microsatellites and faster fixation of newly inserted transposable elements (TEs) on the Y chromosomes. In addition, the X and Y chromosomes, despite similar total counts of TEs, differ significantly in the representation of individual TE lineages, which indicates that transposons proliferate preferentially in either the paternal or the maternal lineage.     

A pronounced evolutionary shift of the pseudoautosomal region boundary in house mice

The pseudoautosomal region (PAR) is essential for the accurate pairing and segregation of the X and Y chromosomes during meiosis. Despite its functional significance, the PAR shows substantial evolutionary divergence in structure and sequence between mammalian species. An instructive example of PAR evolution is the house mouse Mus musculus domesticus (represented by the C57BL/6J strain), which has the smallest PAR among those that have been mapped. In C57BL/6J, the PAR boundary is located just ~700 kb from the distal end of the X chromosome, whereas the boundary is found at a more proximal position in Mus spretus, a species that diverged from house mice 2-4 million years ago. In this study we used a combination of genetic and physical mapping to document a pronounced shift in the PAR boundary in a second house mouse subspecies, Mus musculus castaneus (represented by the CAST/EiJ strain), ~430 kb proximal of the M. m. domesticus boundary. We demonstrate molecular evolutionary consequences of this shift, including a marked lineage-specific increase in sequence divergence within Mid1, a gene that resides entirely within the M. m. castaneus PAR but straddles the boundary in other subspecies. Our results extend observations of structural divergence in the PAR to closely related subspecies, pointing to major evolutionary changes in this functionally important genomic region over a short time period.     

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.     

Chromosome assignment of two cloned DNA probes hybridizing predominantly to human sex chromosomes

Summary In situ hybridization experiments were carried out with two clones, YACG 35 and 2.8, which had been selected from two genomic libraries strongly enriched for the human Y chromosome. Besides the human Y chromosome, both sequences strongly hybridized to the human X chromosome, with few minor binding sites on autosomes. In particular, on the X chromosome DNA from clone YACG 35 hybridized to the centromeric region and the distal part of the short arm (Xp2.2). On the Y chromosome, the sequence was assigned to one site situated in the border region between Yq1.1 and Yq1.2. DNA from clone 2.8 also hybridized to the centromeric region of the X and the distal part of the short arm (Xq2.2). On the Y, however, two binding sites were observed (Yp1.1 and Yq1.2). The findings indicate that sex chromosomal sequences may be localized in homologous regions (as suggested from meiotic pairing) but also at ectopic sites.     

A high frequency of XO offspring from X(Paf)Y* male mice: evidence that the Paf mutation involves an inversion spanning the X PAR boundary

It has previously been reported that 19% of the daughters of males carrying the X-linked mutation patchy fur (Paf) are XO with a maternally derived X chromosome. We now report that hemizygous Paf males that also carry the variant Y chromosome Y*, show a much increased XO production ( approximately 40% of daughters). We hypothesize that the Paf mutation is associated with an inversion spanning the pseudoautosomal region (PAR) boundary, and that this leads to preferential crossing over between the resulting inverted region of PAR and an equivalent inverted PAR region within the compound Y* PAR. This would lead to the production of dicentric X and acentric Y products and consequent sex chromosome loss. This interpretation is supported by analysis of the sex chromosome complements at the second meiotic metaphase, which revealed a high incidence of dicentrics. Another curious feature of the Paf mutation is that mice that are homozygous Paf have more hair than mice that are hemizygous Paf. This can be explained if the Paf mutation is a hypomorphic mutation that escapes X inactivation because, unlike the wild type allele, it is now located within the PAR.     

Concerted evolution of the tandem array encoding primate U2 snRNA occurs in situ, without changing the cytological context of the RNU2 locus.

In primates, the tandemly repeated genes encoding U2 small nuclear RNA evolve concertedly, i.e. the sequence of the U2 repeat unit is essentially homogeneous within each species but differs somewhat between species. Using chromosome painting and the NGFR gene as an outside marker, we show that the U2 tandem array (RNU2) has remained at the same chromosomal locus (equivalent to human 17q21) through multiple speciation events over > 35 million years leading to the Old World monkey and hominoid lineages. The data suggest that the U2 tandem repeat, once established in the primate lineage, contained sequence elements favoring perpetuation and concerted evolution of the array in situ, despite a pericentric inversion in chimpanzee, a reciprocal translocation in gorilla and a paracentric inversion in orang utan. Comparison of the 11 kb U2 repeat unit found in baboon and other Old World monkeys with the 6 kb U2 repeat unit in humans and other hominids revealed that an ancestral U2 repeat unit was expanded by insertion of a 5 kb retrovirus bearing 1 kb long terminal repeats (LTRs). Subsequent excision of the provirus by homologous recombination between the LTRs generated a 6 kb U2 repeat unit containing a solo LTR. Remarkably, both junctions between the human U2 tandem array and flanking chromosomal DNA at 17q21 fall within the solo LTR sequence, suggesting a role for the LTR in the origin or maintenance of the primate U2 array.     

Extensive sequence homologies between Y and other human chromosomes

Twenty-six human Y-chromosome-derived DNA sequences, free of repetitive material, were used to probe male and female genomic blots. We present data from a detailed analysis and chromosomal location of the bands detected by such probes, which demonstrate extensive DNA sequence homology between the mammalian sex chromosomes and autosomes. Under stringent conditions, nine Y-derived probes reacted exclusively with the Y chromosome, 12 probes detected homologous sequences present on both the Y and the X, four probes detected homologies between Y and autosome(s) without any X counterpart and, finally, one probe hybridized to homologous sequences on Y, X and autosome(s). These data are consistent with the hypothesis of a common evolutionary origin for the mammalian sex chromosomes and reveal structural similarities between Y-located and autosomal non-repetitive sequences.     

High-density comparative BAC mapping in the black muntjac (Muntiacus crinifrons): molecular cytogenetic dissection of the origin of MCR 1p+4 in the X1X2Y1Y2Y3 sex chromosome system

The black muntjac (Muntiacus crinifrons, 2n = 8[female symbol]/9[male symbol]) is a critically endangered mammalian species that is confined to a narrow region of southeastern China. Male black muntjacs have an astonishing X1X2Y1Y2Y3 sex chromosome system, unparalleled in eutherian mammals, involving approximately half of the entire genome. A high-resolution comparative map between the black muntjac (M. crinifrons) and the Chinese muntjac (M. reevesi, 2n = 46) has been constructed based on the chromosomal localization of 304 clones from a genomic BAC (bacterial artificial chromosome) library of the Indian muntjac (M. muntjak vaginalis, 2n = 6[female symbol]/7[male symbol]). In addition to validating the chromosomal homologies between M. reevesi and M. crinifrons defined previously by chromosome painting, the comparative BAC map demonstrates that all tandem fusions that have occurred in the karyotypic evolution of M. crinifrons are centromere-telomere fusions. The map also allows for a more detailed reconstruction of the chromosomal rearrangements leading to this unique and complex sex chromosome system. Furthermore, we have identified 46 BAC clones that could be used to study the molecular evolution of the unique sex chromosomes of the male black muntjacs.     

Phylogenetic analysis of a retroposon family as represented on the human X chromosome

SINE-R elements constitute a class of retroposons derived from the long terminal repeat (LTR) of the human endogenous retrovirus HERV-K family that are present in hominoid primates and active in the human genome. In an investigation of the X chromosome, we identified twenty-five SINE-R elements with between 89.6 and 97.7% homology with the SINE-R.C2 element that is human specific, originally identified in the gene for the C2 component of complement. SINE-R.C2 and a sequence HS307 that we previously identified in a region of Xq21.3 that has a recently created homology with a 4 Mb block in Yp11.2 are amongst the group of elements that have diverged furthest from the parent HERV-K10 sequence. The sequence on the X chromosome resemble those that we previously described on chromosomes 7 and 17 and the Y chromosome, with a similar range of variation. Phylogenetic analysis from the retroposon family including those of African great apes using the neighbor-joining method suggests that the SINE-R retroposon family have evolved independently during primate evolution. Further investigation of SINE-R elements on the sex chromosomes, particularly in recently created regions of X-Y homology, may cast light on the timing of the retroposition process and its possible relevance to recent evolutionary change.     

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.     

Molecular differentiation of the homomorphic sex chromosomes inMegaselia scalaris (Diptera) detected by random DNA probes

Randomly cloned DNA fragments and a poly-(GATA) containing sequence were used as probes to identify sex chromosomal inheritance and to detect differences at the molecular level between the homomorphic X and Y in the phorid fly,Megaselia scalaris. Restriction fragment length differences between males and females and between two laboratory stocks of different geographic origin were used to differentiate between sex chromosomal and autosomal origin of the respective fragments. Five random probes detected X and Y chromosomal DNA loci and two others recognized autosomal DNA loci. One random probe and the poly(GATA) probe hybridized with both sex chromosomal and autosomal restriction fragments. Most of the Y chromosomal restriction fragments were conserved in length between the two stocks while most of the X chromosomal and autosomal fragments showed length polymorphism. It was concluded, therefore, that the Y chromosome contains a conserved segment in which crossover is suppressed and restriction site differences have accumulated relative to the X. These chromosomes, therefore, conform to a theoretically expected early stage of sex chromosome evolution.     

Co-amplification of L1 line elements with localised low copy repeats in Giemsa dark bands: implications for genome organisation.

A repeat sequence island, located at the A3 Giemsa dark band on the mouse X chromosome and consisting of 50 copies of a localised long complex repeat unit (LCRU), features an unusually high concentration of L1 LINE repeat sequences juxtaposed and inserted within the LCRU. Sequence analysis of three independent genomic clones containing L1 LINE elements juxtaposed with the LCRU demonstrates a common junction sequence at the L1/LCRU boundary, suggesting that the high concentration of L1 LINE sequences in the repeat sequence island has arisen by association of an L1 element with an LCRU followed by amplification. The LCRU target site at this common junction sequence bears no resemblance to the target site of an L1 element inserted within one LCRU, indicating there is no specific preferential target site for L1 integration. We propose that co-amplification of L1 LINE elements with localised low copy repeat families throughout the genome could have a major effect on the chromosomal distribution of L1 LINE elements.     

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.     

Low X/Y divergence in four pairs of papaya sex-linked genes

Sex chromosomes in flowering plants, in contrast to those in animals, evolved relatively recently and only a few are heteromorphic. The homomorphic sex chromosomes of papaya show features of incipient sex chromosome evolution. We investigated the features of paired X- and Y-specific bacterial artificial chromosomes (BACs), and estimated the time of divergence in four pairs of sex-linked genes. We report the results of a comparative analysis of long contiguous genomic DNA sequences between the X and hermaphrodite Y (Y(h)) chromosomes. Numerous chromosomal rearrangements were detected in the male-specific region of the Y chromosome (MSY), including inversions, deletions, insertions, duplications and translocations, showing the dynamic evolutionary process on the MSY after recombination ceased. DNA sequence expansion was documented in the two regions of the MSY, demonstrating that the cytologically homomorphic sex chromosomes are heteromorphic at the molecular level. Analysis of sequence divergence between four X and Y(h) gene pairs resulted in a estimated age of divergence of between 0.5 and 2.2 million years, supporting a recent origin of the papaya sex chromosomes. Our findings indicate that sex chromosomes did not evolve at the family level in Caricaceae, and reinforce the theory that sex chromosomes evolve at the species level in some lineages.     

The role of chromosomal rearrangements in the evolution of <Emphasis Type="Italic">Silene latifolia</Emphasis> sex chromosomes

Silene latifolia is a model plant for studies of the early steps of sex chromosome evolution. In comparison to mammalian sex chromosomes that evolved 300 mya, sex chromosomes of S. latifolia appeared approximately 20 mya. Here, we combine results from physical mapping of sex-linked genes using polymerase chain reaction on microdissected arms of the S. latifolia X chromosome, and fluorescence in situ hybridization analysis of a new cytogenetic marker, Silene tandem repeat accumulated on the Y chromosome. The data are interpreted in the light of current genetic linkage maps of the X chromosome and a physical map of the Y chromosome. Our results identify the position of the centromere relative to the mapped genes on the X chromosome. We suggest that the evolution of the S. latifolia Y chromosome has been accompanied by at least one paracentric and one pericentric inversion. These results indicate that large chromosomal rearrangements have played an important role in Y chromosome evolution in S. latifolia and that chromosomal rearrangements are an integral part of sex chromosome evolution.     

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.