Replication timing properties within the mouse distal chromosome 7 imprinting cluster

Genomic imprinting is characterized by allele-specific expression of genes within chromosomal domains. Here we show, using fluorescence in situ hybridization (FISH) analysis, that the large chromosomal domain of the mouse distal chromosome 7 imprinting cluster, approximately 1 Mb in length between p57Kip2 and H19 genes, replicates asynchronously between the two alleles during S-phase. At the telomeric side of this domain, we found a transition from asynchronous replication at the imprinted p57Kip2 gene to synchronous replication at the Nap2 gene. Two-color FISH suggested that the paternal allele of this whole domain replicates earlier than its maternal allele. Treatment of the cells with a histone deacetylase inhibitor abolished this allele-specific feature accompanied with accelerated replication of the later-replicating allele at a domain level. Allele-specific asynchronous replication was observed even in ES cells. These results suggest that this imprinting cluster consists of a large replication domain which is already found at the early stage in development.     

Recombination, GC-content and the human pseudoautosomal boundary paradox

The pseudoautosomal boundary of mammalian sex chromosomes separates a low-recombination region (X- or Y-specific) from a high-recombination region (the pseudoautosomal region), providing a good opportunity to investigate the influence of recombination on molecular evolutionary processes. The mouse and human patterns of sequence variation, however, are discordant: a striking difference of GC-content and evolutionary rate was reported between the proximal and distal sides of the pseudoautosomal boundary in the mouse genome, whereas this difference was not found in the human genome. The paradox might be explained by the mirror histories of the pseudoautosomal boundary in the two species, and by the asymmetric nature of the forces driving GC-content evolution in mammalian genomes.     

Physical mapping of 14 new DNA markers isolated from the human distal Xp region.

We have isolated 14 new DNA markers from the human Xpter-Xp21 region distal to the Duchenne muscular dystrophy gene by targeted cloning, employing two somatic cell hybrids containing this region as their sole human material. High-resolution physical localization of these markers within this region was obtained by hybridization to two mapping panels consisting of DNA from patients carrying various translocations and deletions in distal Xp. Five markers were assigned to the pseudoautosomal region where their position on the long-range map of this region was further determined by pulsed-field gel electrophoresis. The other nine markers map to the X-specific region. Informative TaqI restriction fragment length polymorphisms were observed for four loci. One of these represents a region-specific low-copy repeated element. These 14 new markers represent useful tools for the understanding of distal Xp deletion and translocation mechanisms and for the positional cloning of disease genes in the region.     

Role of the pseudoautosomal region in sex-chromosome pairing during male meiosis: meiotic studies in a man with a deletion of distal Xp.

Meiotic studies were undertaken in a 24-year-old male patient with short stature, chondrodysplasia punctata, ichthyosis, steroid sulfatase deficiency, and mild mental retardation with an inherited cytologically visible deletion of distal Xp. Molecular investigations showed that the pseudoautosomal region as well as the steroid sulfatase gene were deleted, but telomeric sequences were present at the pter on the deleted X chromosome. A complete failure of sex-chromosome pairing was observed in the primary spermatocytes of the patient. Telomeric approaches between the sex chromosomes were made at zygotene in some cells, but no XY synaptonemal complex was formed. The sex chromosomes were present as univalents at metaphase I, and germ-cell development was arrested between metaphase I and metaphase II in the vast majority of cells, consistent with the azoospermia observed in the patient. The failure of XY pairing in this individual indicates that the pseudoautosomal sequences play an important role in initiating XY pairing and formation of synaptonemal complex at meiosis.     

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.     

A physical map of the human pseudoautosomal region.

A physical map of the human pseudoautosomal region has been constructed using pulsed field gel electrophoresis and the infrequently cutting restriction enzymes BssHIII, EagI, SstII, NotI, MluI and NruI. This map extends 2.3 Mbp from the telomere to sex-chromosome-specific DNA, includes at least seven CpG islands and locates four genetically mapped loci. Five of the CpG islands are organized into two clusters. One cluster is adjacent to the telomere, the other extends into sex-chromosome-specific DNA. There is congruence between the genetic and physical maps which implies that the frequency of recombination is approximately uniform throughout the DNA.     

A gradient of silent substitution rate in the human pseudoautosomal region

It has been demonstrated that recombination in the human p-arm pseudoautosomal region (p-PAR) is at least twenty times more frequent than the genomic average of approximately 1 cM/Mb, which may affect substitution patterns and rates in this region. Here I report the analysis of substitution patterns and rates in 10 human, chimpanzee, gorilla, and orangutan genes across the p-PAR. Between species silent divergence in the p-PAR forms a gradient, increasing toward the telomere. The correlation of silent divergence with distance from the p-PAR boundary is highly significant (rho = 0.911, P < 0.001). After exclusion of the CpG dinucleotides this correlation is still significant (rho = 0.89, P < 0.01), thus the substitution rate gradient cannot be explained solely by the differences in the extent of methylation across the p-PAR. Frequent recombination in the PAR may result in a relatively strong effect of biased gene conversion (BGC), which, because of the increased probability of fixation of the G or C nucleotides at (A or T)/(G or C) segregating sites, may affect substitution rates. BGC, however, does not seem to be the factor creating the substitution rate gradient in the p-PAR, because the only gradient is still detactable if only A<-->T and G<-->C substitutions are taken into account (rho = 0.82, P < 0.01). I hypothesize that the substitution rate gradient in the p-PAR is due to the mutagenic effect of recombination, which is very frequent in the distal human p-PAR and might be lower near the p-PAR boundary.     

Replication timing of the human X-inactivation center (XIC) region: correlation with chromosome bands

The human genome is composed of long-range G+C% mosaic structures, which are thought to be related to chromosome bands. Replication timing during S phase is associated with chromosomal band zones; thus, band boundaries are thought to correspond to regions where replication timing switches. The proximal limit of the human X-inactivation center (XIC) has been localized cytologically to the junction zone between Xq13.1 and Xq13.2. Using PCR-based quantification of the newly replicated DNA from cell-cycle fractionated THP-1 cells, the replication timing in and around the XIC was determined at the genome sequence level. We found two regions where replication timing changes from the early to late period during S phase. One is located near a large inverted duplication proximal to the XIC, and the other is near the XIST locus. We propose that the 1Mb late-replicated zone (from the large inverted duplication to XIST) corresponds to a G-band Xq13.2. Several common characteristics were observed in the XIST region and the MHC class II-III junction which was previously defined as a band boundary. These characteristics included differential high-density clustering of Alu and LINE repeats, and the presence of polypurine/polypyrimidine tracts, MER41A, MER57 and MER58B.     

Two highly polymorphic minisatellites from the pseudoautosomal region of the human sex chromosomes.

Two pseudoautosomal loci DXYS15 and DXYS17 from the pairing region of the human sex chromosomes display a high variability with at least eight alleles each. The structural elements responsible for the polymorphisms have been isolated and sequenced. In both cases the variations result from DNA rearrangements occurring in tandemly repeated sequences (minisatellites) of 21-29 nucleotides for DXYS15 and 28-33 nucleotides for DXYS17. At reduced stringency, the DXYS15 minisatellite detects other hypervariable sequences located in other parts of the genome and hence represents a new family of minisatellites. In contrast to most other known hypervariable families, the DXYS15 hypervariable sequence displays a very high AT content.     

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.     

Regional mapping of a liver alpha-subunit gene of phosphorylase kinase (PHKA) to the distal region of human chromosome Xp.

X-linked liver glycogenosis (XLG) is a glycogen storage disorder resulting from deficient activity of phosphorylase kinase (PHK). PHK consists of four different subunits: alpha, beta, gamma, and delta. Several genes encoding PHK subunits have been cloned and localized, but only the muscle alpha-subunit (PHKA) gene has been assigned to the X chromosome, in the region Xq12----q13. However, we have previously excluded the muscle PHKA gene as a candidate gene for the XLG mutation, as linkage analysis indicated that the mutation responsible for XLG is located in Xp22 and not in Xq12----q13. We report here the chromosomal localization by in situ hybridization of a liver PHKA gene to the distal region of chromosome Xp. Strong hybridization signals were observed on the distal part of the short arm of a chromosome identified as the X chromosome by cohybridization with an X chromosome-specific centromeric probe. The localization of this gene in the same chromosomal region as the disease gene responsible for XLG suggests that the liver PHKA gene is a highly likely candidate gene for the XLG mutation.     

Replication timing of extremely large genes on human chromosomes 11q and 21q

Many human genes have been mapped precisely in the genome. These genes vary from a few kb to more than 1 Mb in length. Previously, we measured replication timing along the entire lengths of human chromosomes 11q and 21q at the sequence level. In the present study, we used the newest information for human chromosomes 11q and 21q to analyze the replication timing of 30 extremely large genes (>250 kb) in two human cell lines (THP-1 and Jurkat). The timing of replication differed between the 5'- and 3'-ends of each of extremely large genes on 11q and 21q, and the time interval between their replication varied among genes of different lengths. The large genes analyzed here included several tissue-specific genes associated with neural diseases and genes encoding cell adhesion molecules: some of these genes had different patterns of replication timing between the two cell lines. The amyloid precursor protein gene (APP), which is associated with familial Alzheimer's disease (AD1), showed the largest difference in timing of replication between its 5'- and 3'-ends in relation to gene length of all the large genes studied on 11q and 21q. These extremely large genes were concentrated in and around genomic regions in which replication timing switches from early to late on both 11q and 21q. The differences of replication timing between the 5'- and 3'-terminal regions of large genes may be related to the molecular mechanisms that underlie tissue-specific expression.     

Replication timing of DNA sequences associated with human centromeres and telomeres.

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.     

Strong linkage disequilibrium between the XY274 polymorphism and the pseudoautosomal boundary.

The pseudoautosomal boundary is defined by an Alu repeat element on the Y chromosome. The Alu element is found on all Y chromosomes and on no X chromosomes, establishing it as part of Y-specific sequences. Distal to the Alu element, sequences from the X and Y are strictly homologous, suggesting that the boundary is formed by an abrupt break in sequence homology. Further investigation of the function of the boundary has been undertaken by examining the population structure of an MspI restriction-site polymorphism (XY274), which is located 274 bp distal to the Alu insertion site. Southern blot and polymerase chain reaction analysis demonstrate fixation of the high allele (noncutting or AT base pair) of XY274 on the Y chromosome in most populations, while a full range of high allele frequencies is found on the X chromosomes of different populations. Two exceptions to fixation on the Y chromosome were found in African populations. The level of linkage disequilibrium suggests that the first few hundred base pairs of the pseudoautosomal region on the Y chromosome share a single common origin more recent than the origin of the species.     

Evolution of the pseudoautosomal boundary in Old World monkeys and great apes

Mammalian sex chromosomes are divided into sex-specific and pseudoautosomal regions. Sequences in the pseudoautosomal region recombine between the sex chromosomes; the sex-specific sequences normally do not. The interface between sex-specific and pseudoautosomal sequences is the pseudoautosomal boundary. The boundary is the centromeric limit to recombination in the pseudoautosomal region. In man, an Alu repeat element is found inserted at the boundary on the Y chromosome. In the evolutionary comparison conducted here, the Alu repeat element is found at the Y boundary in great apes, but it is not found there in two Old World monkeys. During the evolution of the Old World monkey and great ape lineages, homology between the sex chromosomes was maintained by recombination in the sequences telomeric to the Alu insertion site. The Alu repeat element did not create the present-day boundary; instead, it inserted at the preexisting boundary after the Old World monkey and great ape lineages diverged.     

Linkage studies do not confirm the cytogenetic location of incontinentia pigmenti on Xp11

Summary Linkage studies have been performed in 5 incontinentia pigmenti (IP) families totaling 29 potentially informative meioses. Ten probes of the Xp arm were used, six of them were precisely localized on the X chromosome, using hamster x human somatic cell hybrids containing a broken X chromosome derived from an incontinentia pigmenti patient carrying an X;9 translocation [46,XX,t(X;9)(p11.21;q34)]. The following order for probes is proposed: pter-(DXS7, DXS146, DXS255)-IP1-(DXS14, DXS90)-DXS106-qter. The negative lod scores obtained exclude the possibility that in the families studied, the gene for IP is located in Xp11 or in the major part of the Xp arm.     

Molecular cytogenetic analysis of a duplication Xp in a male: further delineation of a possible sex influencing region on the X chromosome

We describe a male infant with severe mental retardation and autism with a duplication of the short arm of the X chromosome. Chromosome painting confirmed the origin of this X duplication. Molecular cytogenetic analysis with fluorescence in situ hybridization (FISH) identified one copy of the zinc finger protein on the X chromosome (ZFX) and two copies of the steroid sulfatase gene (STS), further delineating the breakpoints. Based on cytogenetic and molecular comparisons of cases from the literature of sex-reversal in dup(X),Y patients and our patient, we suggest that a possible secondary sexinfluencing gene involved in the regulation of sex determination or testis morphogenesis is present at the distal Xp21.1 to p21.2 region.     

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.     

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.     

FISH-deletion mapping defines a 270-kb short stature critical interval in the pseudoautosomal region PAR1 on human sex chromosomes

Deletions of the pseudoautosomal region (PAR1) of the sex chromosomes have recently been discovered in individuals with short stature, and a minimal common deletion region of 700 kb within PAR1 has subsequently been defined. We have cloned this entire region, which is bounded by the Xp/Yp telomere, as an overlapping cosmid contig. In the present study, we have used fluorescence in situ hybridization (FISH) to study four patients with X-chromosomal rearrangements, two with normal height and two with short stature. Genotype-phenotype correlations have narrowed down the the critical “short stature interval” to a 270-kb region containing the gene with an important role in growth. A minimal tiling path of 6–8 cosmids bridging this interval is now available for interphase and metaphase FISH and provides a valuable tool for diagnostic investigations of patients with idiopathic short stature. Received: 4 November 1996 / Accepted: 10 March 1997