Evolutionary molecular cytogenetics of catarrhine primates: past, present and future.

The catarrhine primates were the first group of species studied with comparative molecular cytogenetics. Many of the fundamental techniques and principles of analysis were initially applied to comparisons in these primates, including interspecific chromosome painting, reciprocal chromosome painting and the extensive use of cloned DNA probes for evolutionary analysis. The definition and importance of chromosome syntenies and associations for a correct cladistics analysis of phylogenomic relationships were first applied to catarrhines. These early chromosome painting studies vividly illustrated a striking conservation of the genome between humans and macaques. Contemporarily, it also revealed profound differences between humans and gibbons, a group of species more closely related to humans, making it clear that chromosome evolution did not follow a molecular clock. Chromosome painting has now been applied to more that 60 primate species and the translocation history has been mapped onto the major taxonomic divisions in the tree of primate evolution. In situ hybridization of cloned DNA probes, primarily BAC-FISH, also made it possible to more precisely map breakpoints with spanning and flanking BACs. These studies established marker order and disclosed intrachromosomal rearrangements. When applied comparatively to a range of primate species, they led to the discovery of evolutionary new centromeres as an important new category of chromosome evolution. BAC-FISH studies are intimately connected to genome sequencing, and probes can usually be assigned to a precise location in the genome assembly. This connection ties molecular cytogenetics securely to genome sequencing, assuring that molecular cytogenetics will continue to have a productive future in the multidisciplinary science of phylogenomics.

Centromere repositioning in mammals.

The evolutionary history of chromosomes can be tracked by the comparative hybridization of large panels of bacterial artificial chromosome clones. This approach has disclosed an unprecedented phenomenon: 'centromere repositioning', that is, the movement of the centromere along the chromosome without marker order variation. The occurrence of evolutionary new centromeres (ENCs) is relatively frequent. In macaque, for instance, 9 out of 20 autosomal centromeres are evolutionarily new; in donkey at least 5 such neocentromeres originated after divergence from the zebra, in less than 1 million years. Recently, orangutan chromosome 9, considered to be heterozygous for a complex rearrangement, was discovered to be an ENC. In humans, in addition to neocentromeres that arise in acentric fragments and result in clinical phenotypes, 8 centromere-repositioning events have been reported. These 'real-time' repositioned centromere-seeding events provide clues to ENC birth and progression. In the present paper, we provide a review of the centromere repositioning. We add new data on the population genetics of the ENC of the orangutan, and describe for the first time an ENC on the X chromosome of squirrel monkeys. Next-generation sequencing technologies have started an unprecedented, flourishing period of rapid whole-genome sequencing. In this context, it is worth noting that these technologies, uncoupled from cytogenetics, would miss all the biological data on evolutionary centromere repositioning. Therefore, we can anticipate that classical and molecular cytogenetics will continue to have a crucial role in the identification of centromere movements. Indeed, all ENCs and human neocentromeres were found following classical and molecular cytogenetic investigations.

Chromosome painting in two genera of South American monkeys: species identification, conservation, and management.

Cytogenetic studies showed that a number of New World primate taxa, particularly the genera Alouatta, Aotus, and Callicebus, have highly derived karyotypes. Cytogenetics in these primates, at every level of analysis, has contributed to the recognition of species and revealed that their number was certainly underestimated by researchers relying solely on traditional morphological data. Further attention was drawn to Alouatta and Aotus because they are characterized by translocations of the Y chromosome to autosomes, generating multiple sex chromosome systems. Here we present a report on the hybridization of human chromosome-specific paints on metaphases from 4 individuals originally assigned to Alouatta caraya and 1 individual of Aotuslemurinus. This is only the third karyotype studied with chromosome painting out of more than 10 known karyomorphs in Aotus. The banded chromosomes matched those of karyotype II as defined by Ma et al. [1976a], and we were able to more precisely assign the origin of the sample to A. l. griseimembra. Our results on the Argentinean Alouatta caraya samples were generally comparable to the banding and hybridization pattern of previous studies of A. caraya including the presence of an X(1)X(1)X(2)X(2)/X(1)X(2)Y(1)Y(2) sex chromosome system. The karyotype of the Brazilian Alouatta sample labeled as A. caraya differs from the 3 Argentinean samples by at least 10 chromosome rearrangements. The diploid number, G banding, and hybridization pattern of this female cell line was almost identical to previous painting results on Alouatta guariba guariba. Therefore we must conclude that this cell line is actually from an A. guariba guariba individual. The contribution of cytogenetic tools in identifying species or in this case assigning individuals or cell lines to their precise taxonomic allocation is stressed. Gathering further molecular cytogenetic data on New World primates should be conservation and management priorities.

Primate chromosome evolution: ancestral karyotypes, marker order and neocentromeres.

In 1992 the Japanese macaque was the first species for which the homology of the entire karyotype was established by cross-species chromosome painting. Today, there are chromosome painting data on more than 50 species of primates. Although chromosome painting is a rapid and economical method for tracking translocations, it has limited utility for revealing intrachromosomal rearrangements. Fortunately, the use of BAC-FISH in the last few years has allowed remarkable progress in determining marker order along primate chromosomes and there are now marker order data on an array of primate species for a good number of chromosomes. These data reveal inversions, but also show that centromeres of many orthologous chromosomes are embedded in different genomic contexts. Even if the mechanisms of neocentromere formation and progression are just beginning to be understood, it is clear that these phenomena had a significant impact on shaping the primate genome and are fundamental to our understanding of genome evolution. In this report we complete and integrate the dataset of BAC-FISH marker order for human syntenies 1, 2, 4, 5, 8, 12, 17, 18, 19, 21, 22 and the X. These results allowed us to develop hypotheses about the content, marker order and centromere position in ancestral karyotypes at five major branching points on the primate evolutionary tree: ancestral primate, ancestral anthropoid, ancestral platyrrhine, ancestral catarrhine and ancestral hominoid. Current models suggest that between-species structural rearrangements are often intimately related to speciation. Comparative primate cytogenetics has become an important tool for elucidating the phylogeny and the taxonomy of primates. It has become increasingly apparent that molecular cytogenetic data in the future can be fruitfully combined with whole-genome assemblies to advance our understanding of primate genome evolution as well as the mechanisms and processes that have led to the origin of the human genome.

Molecular evolution of the human chromosome 15 pericentromeric region.

We present a detailed molecular evolutionary analysis of 1.2 Mb from the pericentromeric region of human 15q11. Sequence analysis indicates the region has been subject to extensive interchromosomal and intrachromosomal duplications during primate evolution. Comparative FISH analyses among non-human primates show remarkable quantitative and qualitative differences in the organization and duplication history of this region - including lineage-specific deletions and duplication expansions. Phylogenetic and comparative analyses reveal that the region is composed of at least 24 distinct segmental duplications or duplicons that have populated the pericentromeric regions of the human genome over the last 40 million years of human evolution. The value of combining both cytogenetic and experimental data in understanding the complex forces which have shaped these regions is discussed.

Using a pericentromeric interspersed repeat to recapitulate the phylogeny and expansion of human centromeric segmental duplications.

Despite considerable advances in sequencing of the human genome over the past few years, the organization and evolution of human pericentromeric regions have been difficult to resolve. This is due, in part, to the presence of large, complex blocks of duplicated genomic sequence at the boundary between centromeric satellite and unique euchromatic DNA. Here, we report the identification and characterization of an approximately 49-kb repeat sequence that exists in more than 40 copies within the human genome. This repeat is specific to highly duplicated pericentromeric regions with multiple copies distributed in an interspersed fashion among a subset of human chromosomes. Using this interspersed repeat (termed PIR4) as a marker of pericentromeric DNA, we recovered and sequence-tagged 3 Mb of pericentromeric DNA from a variety of human chromosomes as well as nonhuman primate genomes. A global evolutionary reconstruction of the dispersal of PIR4 sequence and analysis of flanking sequence supports a model in which pericentromeric duplications initiated before the separation of the great ape species (>12 MYA). Further, analyses of this duplication and associated flanking duplications narrow the major burst of pericentromeric duplication activity to a time just before the divergence of the African great ape and human species (5 to 7 MYA). These recent duplication exchange events substantially restructured the pericentromeric regions of hominoid chromosomes and created an architecture where large blocks of sequence are shared among nonhomologous chromosomes. This report provides the first global view of the series of historical events that have reshaped human pericentromeric regions over recent evolutionary time.

Human genome dispersal and evolution of 4q35 duplications and interspersed LSau repeats.

We have investigated the evolutionary history of the 4q35 paralogous region, and of a sub-family of interspersed LSau repeats. In HSA, 4q35 duplications were localized at 1q12, 3p12.3, 4q35, 10q26, 20cen, whereas duplicons and interspersed LSau repeats simultaneously labeled the p arm of acrocentric chromosomes. A multi-site localization of 4q35-like sequences was also observed in PTR, GGO, PPY, HLA (Hominoidea) and PAN (Old World monkey), thus indicating that duplications of this region have occurred extensively in the two clades, which diverged at least 25 million years ago. In HSA, PTR and PAN, 4q35-derived duplicons co-localized with rDNA, whereas in GGO and PPY this association was partially lacking. In PAN, the single- and multi-site distribution of rDNA and paralogous sequences, respectively, indicates a different timing of sequence dispersal. The sub-family of interspersed LSau repeats showed a lesser dispersal than 4q35 duplications both in man and great apes. This finding suggests that duplications and repeated sequences have undergone different expansion/contraction events during evolution. The mechanisms underlying the dispersal of paralogous regions may be further derived through studies comparing the detailed structural organization of these genomic regions in man and primates.

Differential divergence of three human pseudoautosomal genes and their mouse homologs: implications for sex chromosome evolution.

The human pseudoautosomal region 1 (PAR1) is essential for meiotic pairing and recombination, and its deletion causes male sterility. Comparative studies of human and mouse pseudoautosomal genes are valuable in charting the evolution of this interesting region, but have been limited by the paucity of genes conserved between the two species. We have cloned a novel human PAR1 gene, DHRSXY, encoding an oxidoreductase of the short-chain dehydrogenase/reductase family, and isolated a mouse ortholog Dhrsxy. We also searched for mouse homologs of recently reported PGPL and TRAMP genes that flank it within PAR1. We recovered a highly conserved mouse ortholog of PGPL by cross-hybridization, but found no mouse homolog of TRAMP. Like Csf2ra and Il3ra, both mouse homologs are autosomal; Pgpl on chromosome 5, and Dhrsxy subtelomeric on chromosome 4. TRAMP, like the human genes within or near PAR1, is probably very divergent or absent in the mouse genome. We interpret the rapid divergence and loss of pseudoautosomal genes in terms of a model of selection for the concentration of repetitive recombinogenic sequences that predispose to high recombination and translocation.

Characterization of a highly repeated DNA sequence family in five species of the genus Eulemur.

The karyotypes of Eulemur species exhibit a high degree of variation, as a consequence of the Robertsonian fusion and/or centromere fission. Centromeric and pericentromeric heterochromatin of eulemurs is constituted by highly repeated DNA sequences (including some telomeric TTAGGG repeats) which have so far been investigated and used for the study of the systematic relationships of the different species of the genus Eulemur. In our study, we have cloned a set of repetitive pericentromeric sequences of five Eulemur species: E. fulvus fulvus (EFU), E. mongoz (EMO), E. macaco (EMA), E. rubriventer (ERU), and E. coronatus (ECO). We have characterized these clones by sequence comparison and by comparative fluorescence in situ hybridization analysis in EMA and EFU. Our results showed a high degree of sequence similarity among Eulemur species, indicating a strong conservation, within the five species, of these pericentromeric highly repeated DNA sequences.

Centromere emergence in evolution.

Evolutionary centromere repositioning is a paradox we have recently discovered while studying the conservation of the phylogenetic chromosome IX in primates. Two explanations were proposed: a conservative hypothesis assuming sequential pericentric inversions, and a more challenging assumption involving centromere emergence during evolution. The complex evolutionary history showed by chromosome IX did not allow us to clearly distinguish between these two hypotheses. Here we report comparative studies of chromosome X in two lemur species: the black lemur and the ringtailed lemur. The X chromosome is telocentric in the black lemur and almost metacentric in the ringtailed lemur. The marker order along these chromosomes, however, was found to be perfectly colinear with humans. Our data unequivocally point to centromere emergence as the most likely explanation of centromere repositioning.

Divergent origins and concerted expansion of two segmental duplications on chromosome 16.

An unexpected finding of the human genome was the large fraction of the genome organized as blocks of interspersed duplicated sequence. We provide a comparative and phylogenetic analysis of a highly duplicated region of 16p12.2, which is composed of at least four different segmental duplications spanning in excess of 160 kb. We contrast the dispersal of two different segmental duplications (LCR16a and LCR16u). LCR16a, a 20 kb low-copy repeat sequence A from chromosome 16, was shown previously to contain a rapidly evolving novel hominoid gene family (morpheus) that had expanded within the last 10 million years of great ape/human evolution. We compare the dispersal of this genomic segment with a second adjacent duplication called LCR16u. The duplication contains a second putative gene family (KIAA0220/SMG1) that is represented approximately eight times within the human genome. A high degree of sequence identity (approximately 98%) was observed among the various copies of LCR16u. Comparative analyses with Old World monkey species show that LCR16a and LCR16u originated from two distinct ancestral loci. Within the human genome, at least 70% of the LCR16u copies were duplicated in concert with the LCR16a duplication. In contrast, only 30% of the chimpanzee loci show an association between LCR16a and LCR16u duplications. The data suggest that the two copies of genomic sequence were brought together during the chimpanzee/human divergence and were subsequently duplicated as a larger cassette specifically within the human lineage. The evolutionary history of these two chromosome-specific duplications supports a model of rapid expansion and evolutionary turnover among the genomes of man and the great apes.

Breakpoint of a Y chromosome pericentric inversion in the DAZ gene area. A case report.

BACKGROUND: The presence of a spermatogenesis locus (gene or gene complex) in the euchromatic region of the long arm of the Y chromosome (Yq11), defined as azoospermia factor on the basis of gross structural rearrangement, was detected. The gene family responsible for different spermatogenetic defects is "deleted in azoospermia" (DAZ). CASE: A 34-year-old man had oligozoospermia, and a cytogenetic analysis carried out on peripheral lymphocytes with G banding revealed a 46,X, inv(Y)(p11q11)karyotype. The relation between the chromosomal breakpoint and the DAZ gene was more precisely defined by a fluorescent in situ hybridization technique. We revealed two signals for the DAZ gene, weaker than normal, one on the short arm and the other on the long arm of the Y chromosome, indicating that the breakpoint was located at the DAZ gene level. CONCLUSION: This is the first report documenting a chromosomal pericentric inversion with disruption in the DAZ gene area. We hope to obtain information on whether the disruption affects a functional zone of the gene and correlates with oligospermia at the chromosomal level.

Genomic sequence and transcriptional profile of the boundary between pericentromeric satellites and genes on human chromosome arm 10q.

The organization of centromeric heterochromatin has been established in a number of eucaryotes but remains poorly defined in human. Here we present 1025 kb of contiguous human genomic sequence which links pericentromeric satellites to the RET proto-oncogene in 10q11.2 and is presumed to span the transition from centric heterochromatin to euchromatin on this chromosome arm. Two distinct domains can be defined within the sequence. The proximal approximately 240 kb consists of arrays of satellites and other tandem repeats separated by tracts of complex sequence which have evolved by pericentromeric-directed duplication. Analysis of 32 human paralogues of these sequences indicates that most terminate at or within repeat arrays, implicating these repeats in the interchromosomal duplication process. Corroborative PCR-based analyses establish a genome-wide correlation between the distribution of these paralogues and the distribution of satellite families present in 10q11. In contrast, the distal approximately 780 kb contains few tandem repeats and is largely chromosome specific. However, a minimum of three independent intrachromosomal duplication events have resulted in >370 kb of this sequence sharing >90% identity with sequences on 10p. Using computer-based analyses and RT-PCR we confirm the presence of three genes within the sequence, ZNF11/33B, KIAA0187 and RET, in addition to five transcripts of unknown structure. All of these transcribed sequences map distal to the satellite arrays. The boundary between satellite-rich interchromosomally duplicated DNA and chromosome-specific DNA therefore appears to define a transition from pericentromeric heterochromatin to euchromatin on the long arm of this chromosome.

Molecular cytogenetic resources for chromosome 4 and comparative analysis of phylogenetic chromosome IV in great apes.

We have generated a panel of 55 somatic cell hybrids retaining fragments of human chromosome 4. Each hybrid has been characterized cytogenetically by FISH and molecularly by 37 STSs, evenly spaced along the chromosome. The panel can be exploited to map subregionally DNA sequences on chromosome 4 and to generate partial chromosome paints useful in the characterization of chromosomal rearrangements involving this chromosome. Furthermore, a panel of 84 YACs mapping on chromosome 4 has been characterized by FISH. A subset of this panel is recognized by STSs used in the somatic cell hybrid characterization. In this way a correlation between the genetic and the physical maps can be established. These resources have been used to investigate the conservation of the phylogenetic chromosome IV in great apes. The results indicate that all the pericentric inversions that differentiate chromosome IV in these species are distinct and that one of the breakpoints frequently lies very close to the centromere. In 4 instances, the YAC containing the breakpoint was identified. The breakpoint in IVq of PTR and MMU lies in the same YAC, suggesting that this breakpoint has been utilized twice in the evolutionary history of this chromosome.

Differentially regulated and evolved genes in the fully sequenced Xq/Yq pseudoautosomal region.

Human sex chromosomes, which are morphologically and genetically different, share few regions of homology. Among them, only pseudoautosomal regions (PARs) pair and recombine during meiosis. To better address the complex biology of these regions, we sequenced the telomeric 400 kb of the long arm of the human X chromosome, including 330 kb of the human Xq/YqPAR and the telomere. Sequencing reveals subregions with distinctive regulatory and evolutionary features. The proximal 295 kb contains two genes inactivated on both the inactive X and Y chromosomes [ SYBL1 and a novel homologue ( HSPRY3 ) of Drosophila sprouty ]. The GC-rich distal 35 kb, added in stages and much later in evolution, contains the X/Y expressed gene IL9R and a novel gene, CXYorf1, only 5 kb from the Xq telomere. These properties make Xq/YqPAR a model for studies of region-specific gene inactivation, telomere evolution, and involvement in sex-limited conditions.

Molecular cytogenetic dissection of human chromosomes 3 and 21 evolution.

Chromosome painting in placental mammalians illustrates that genome evolution is marked by chromosomal synteny conservation and that the association of chromosomes 3 and 21 may be the largest widely conserved syntenic block known for mammals. We studied intrachromosomal rearrangements of the syntenic block 3/21 by using probes derived from chromosomal subregions with a resolution of up to 10-15 Mbp. We demonstrate that the rearrangements visualized by chromosome painting, mostly translocations, are only a fraction of the actual chromosomal changes that have occurred during evolution. The ancestral segment order for both primates and carnivores is still found in some species in both orders. From the ancestral primate/carnivore condition an inversion is needed to derive the pig homolog, and a fission of chromosome 21 and a pericentric inversion is needed to derive the Bornean orangutan condition. Two overlapping inversions in the chromosome 3 homolog then would lead to the chromosome form found in humans and African apes. This reconstruction of the origin of human chromosome 3 contrasts with the generally accepted scenario derived from chromosome banding in which it was proposed that only one pericentric inversion was needed. From the ancestral form for Old World primates (now found in the Bornean orangutan) a pericentric inversion and centromere shift leads to the chromosome ancestral for all Old World monkeys. Intrachromosomal rearrangements, as shown here, make up a set of potentially plentiful and informative markers that can be used for phylogenetic reconstruction and a more refined comparative mapping of the genome.

Molecular structure and evolution of an alpha satellite/non-alpha satellite junction at 16p11.

We have determined the detailed molecular structure and evolution of an alpha satellite junction from human chromosome 16p11. The analysis reveals that the alpha satellite sequence bordering the transition lacks higher-order structure and that the non-alpha satellite portion consists of a mosaic of duplicated segments of complex evolutionary origin. The 16p11 junction was formed recently (5-10 million years ago) by the duplication and transposition of genomic segments from Xq28 and 4q24. Once this mosaic structure was formed, a larger complex was spread among multiple pericentromeric regions. This resulted in the formation of large (>62 kb) paralogous segments that share a high degree ( approximately 97%) of sequence similarity. Both phylogenetic and comparative analyses indicate that these pericentromeric-directed duplications occurred around the time of the divergence of the human, gorilla and chimpanzee lineages, resulting in the subtle restructuring of the primate genome among these species. The available data suggest that such chimeric structures are a general property of several different human chromosomes near their alpha satellite junctions.

Evolution of the X-specific block embedded in the human Xq21.3/Yp11.1 homology region.

The region Xq21.3/Yp11.1 represents the largest segment of homology between the sex chromosomes in humans, though no recombination occurs in male meiosis. It presumably arose as a transposition from the X to the Y chromosome; the present-day organization in the latter chromosome indicates a paracentric inversion that disrupted its continuity. Moreover, an X-specific block (defined by the marker DXS214) is embedded in the region. Previously, no hypotheses about the length, origin, or evolution of this X-specific segment have been proposed. Here we report on the refinement of the size and the sequence of the distal boundary of the X-specific block. Furthermore, we have tracked by FISH experiments the evolution of this region in primates. This further clarifies the multistep mechanism of origin for the XY homology region, by demonstrating that the X-specific block was deleted from the Y chromosome after the initial transfer from the X chromosome.

Centromere repositioning.

Primate pericentromeric regions recently have been shown to exhibit extraordinary evolutionary plasticity. In this paper we report an additional peculiar feature of these regions that we discovered while analyzing, by FISH, the evolutionary conservation of primate phylogenetic chromosome IX. If the position of the centromere is not taken into account, a relatively small number of rearrangements must be invoked to account for interspecific differences. Conversely, if the centromere is included, a paradox emerges: The position of the centromere seems to have undergone, in some species, an evolutionary history independent from the surrounding markers. A significant number of additional rearrangements must be proposed to reconcile the order of the markers with centromere position. Alternatively, the evolutionary emergence of neocentromeres can be postulated.