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ILAR Journal V39(2/3) 1998
Comparative Gene Mapping
Johannes Wienberg and Roscoe Stanyon
| Johannes Wienberg, Ph.D., is a visiting scientist in the Department of Pathology, University of Cambridge, Cambridge, United Kingdom, and Associate Professor in Human Genetics and Anthropology at the Institute of Anthropology and Human Genetics, München, Germany. Roscoe Stanyon, Ph.D., is Associate Professor in Anthropology in the Department of Anthropological Sciences, University of Genoa, Genoa, Italy. Both authors recently moved to the Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland. |
INTRODUCTION
Since the 1970s, comparative maps for approximately 50 different vertebrate species, chiefly mammals that serve as biomedical models or economically important farm animals, have been constructed using various methods (CGOW 1996). Because of their similarity to humans considerable efforts have also been made to construct maps from primates including macaques, baboons, African green monkeys, various New World monkeys, and prosimians. The principal techniques included pedigree analysis, somatic cell hybrid panels, and cytogenetic studies of chromosome banding patterns. Comparisons of cat, cattle, mouse, pig, and other species suggested that long stretches of syntenic homology existed with the human genome; however, large segments of their genomes remained uncharted. The density of mapped coding genes in almost all species was low, and only a few genes were mapped in 2 or more species. Exceptionally in the mouse, thousands of genes have been mapped, many are already cloned, and more than 1900 genes have been mapped in both human and mouse (Blake and others 1997; http://www.informatics.jax.org). However, in the mouse and rat--the most important animal models in biomedical research--only about 430 genes were assigned to specific chromosomes in both species (CGOW 1996; Levan and others 1991; MGD 1998; Szpirer and others 1996). Gene mapping in most other animals has lagged far behind.
Charting the genome and mapping syntenic homology became more rapid and economical with the introduction of fluorescence in situ hybridization (FISH1) of human specific DNA probes to nonhuman chromosomes. As a result of the human genome project, DNA probes of various complexities are now available to the scientific community and can be readily used for comparative genome analysis. At the time of this writing, the most attractive probes are chromosome paints, which are labeled mixtures of DNA sequences usually derived from flow-sorted or microdissected chromosomes. The paint, which is specific for a single chromosome, hybridizes to the entire chromosome or chromosome segments in a target metaphase. Then the hybridization is detected with fluorescent antibodies against the probe label (Cremer and others 1988; Lichter and others 1988; Pinkel and others 1988).
In addition to probes specific for entire chromosomes, there are subchromosomal and even chromosome band-specific probes derived from rearranged somatic cell hybrids or by microdissection of chromosome regions, in addition, inserts of cloned DNA fragments of various sizes, as well as single genes, are readily available. The various probes have different resolutions that analogous to geographical maps, delineate entire continents, countries, or even villages, streets and houses. All of these probes will certainly contribute to a detailed understanding of genome reshuffling. Chromosome-specific painting probes now have begun to provide an outline of genome evolution in mammals and especially in primates.
Analysis of highly rearranged gibbon karyotypes (see below) allows us to estimate the resolution of chromosome painting in closely related species. Translocations the size of only 1 chromosome band (5 to 10 Mbp) could be visualized by this technique. However, it is obvious from chromosome banding studies that various intrachromosomal rearrangements in primate karyotypes (inversions, transpositions) cannot always be analyzed with chromosome-specific painting probes. Yet these rearrangements can be defined by subregional probes (Arnold and others 1995; Gläser and others 1997; Haaf and Bray-Ward 1996; Lengauer and others 1991; Müller and others 1996; Ried and others 1993; Wienberg and Stanyon 1995, 1997), reciprocal paints derived from highly rearranged karyotypes (Arnold and others 1996, Müller and others 1998), or composite molecular probes mimicking chromosome banding patterns (Müller and others 1997b).
Developments in FISH technology have dramatically increased the use of these techniques both for clinical cytogenetics and basic research. Fluorescence labeled DNA probes allow the differential detection of multiple probes in a single hybridization experiment (Nederlof and others 1989, 1990, 1992; Ried and others 1992). Hybridization images are now routinely recorded by sensitive charge coupled device ("CCD") cameras. After the images are recorded by digital techniques in black and white or pseudocolored formats, the images are merged (Figure 1). Recent advances make it possible to discriminate the entire human chromosome set with 24 differently colored chromosomes in a single experiment (Figure 2) and to define chromosome rearrangements for the entire genome with ease (Schröck and others 1996; Speicher and others 1996).
Since 1990, researchers using human chromosome specific libraries and chromosome paints (Wienberg and others 1990) have established at the DNA level the homology between chromosomes or chromosome subregions of a good number of primates. This technique has been increasingly used to compare the human genome with that of even more distant mammals (Ferguson-Smith and others 1998; Scherthan and others 1994; Wienberg and Stanyon 1995, 1997). Changes in mammalian genomes that occurred during evolution have been mapped virtually by direct observation under the microscope using molecularly defined DNA probes.
Within primates, approximately 20 species have been analyzed by chromosome painting including most major taxonomic divisions. This article summarizes these chromosome painting results. Although this survey is far from complete, the data available at the time of this writing allow some hypotheses to be developed about the origin and evolution of the primate genome.
USING CHROMOSOME DATA IN THE UNDERSTANDING OF PHYLOGENIES AND THE EVOLUTION OF GENOMES
Traditionally, chromosome morphology provided an important tool to study the evolutionary process of genomic changes; however, classical chromosome banding techniques lacked the power to establish homology or locate rearrangement breakpoints with precision. Fortunately, molecular probes now provide this precision. Chromosome painting in highly rearranged genomes such as those of gibbons, many New World primates, and prosimians has shown clearly that previous chromosomal homologies established on the basis of banding or even gene mapping were not reliable. Cytogenetic or chromosomal phylogenies for animal species must be based on homologies established on the basis of DNA content and not morphological similarity.
To distinguish ancestral (symplesiomorphic) from derived (apomorphic) chromosomal arrangements, appropriate "outgroup" species are necessary. Ancestral chromosomal syntenies can then be identified by their presence in the outgroup species and in the species analyzed. Chromosome rearrangements are believed to be rare, and the same rearrangements in different phylogenetic lines are taken to indicate a common evolutionary origin. Common derived (synapomorphic) rearrangements provide the information to link species phylogenetically. Our use of nonprimate mammals as outgroups allowed us to identify the direction of changes within primates. Indeed, FISH using human chromosome-specific painting probes has provided substantial data on interchromosomal rearrangements (translocations) in a number of species, permitting hypotheses of chromosomal syntenies present at each major branching point in primate evolution.
CHROMOSOME PAINTING IN VARIOUS PRIMATES
Great Apes
Great apes, including the common chimpanzee (Pan troglodytes), lowland gorilla (Gorilla gorilla), and the Sumatran subspecies of the orangutan (Pongo pygmaeus abelii), were painted with human chromosome-specific probes (Jauch and others 1992; Stanyon and others 1992; Wienberg and others 1990). Our unpublished data also include the pygmy chimpanzee (Pan paniscus) and the Bornean subspecies of the orangutan (Pongo pygmaeus pygmaeus). As expected from various chromosome banding studies, the human chromosome 2 painting probe hybridized to 2 pairs of homologous chromosomes in all great apes. Two pairs of homologues are ancestral, because this condition is also found in all other higher primates (Bigoni and others 1997a,b; P. FineIli and others, Cambridge University, personal communication, 1998; Wienberg and others 1992).
Recently, painting probes derived by microdissection of the short and long arm of human chromosome 2 were used to delineate the homologous chromosomes in great apes and Old World monkeys (Arnold and others 1996; Wienberg and others 1994). Interestingly, these probes marked the proposed fusion point of the 2 ancestral chromosomes close to band 2q12. This proposal has been confirmed by other probes close to the assumed fusion point including cosmids derived by V kappa immunoglobulin gene probes (Arnold and others 1995) and yeast artificial chromosomes (YACs1) (Haaf and Bray-Ward 1997). Additional evidence for the delineation of the fusion point came from the molecular cytogenetic analyses of even more informative probes mapping to this region. A relict highly repetitive centromere-specific (alphoid) sequence was found distal to the assumed fusion point in 2q21 by in situ hybridization under low hybridization stringency (Avarello and others 1992; Baldini and others 1992). This alphoid sequence may indicate the ancestral centromere of 1 of the fused chromosomes. Furthermore, proximal to 2q21, telomeric sequences were found in band 2ql 3 by in situ hybridization of DNA probes containing inverted arrays of the vertebrate telomeric repeat in a head-to-head arrangement (Ijdo and others 1991; Wells and others 1990). Therefore, this band should represent the ancestral fusion point leading to modern human chromosome 2 (Figure 3).
Analysis of chromosome banding patterns in humans and great apes appeared to indicate that a number of chromosome rearrangements hypothesized to explain differences between these species were informative about human origins and phylogeny (Marks 1992; Stanyon and Chiarelli 1983). For instance, humans and chimpanzees appeared to be phylogenetically linked by a common derived inversion in a homologue to human 2p. This inversion was hypothesized to have occurred in the last common ancestor for both species after divergence from the gorilla. Apparently, both the gorilla and the orangutan conserved the ancestral forms (Dutrillaux 1975; Yunis and Prakash 1982). Indeed, the hybridization pattern of a painting probe for human 2p on great ape chromosomes seems to support this conclusion (Wienberg and others 1995). However, the ancestral form of this chromosome had not been well established. Additional analysis of the homologous chromosomes in an appropriate outgroup, represented by Old World monkeys (Macaca fuscata, Cercopithecus aethiops) revealed a hybridization pattern like that found in the chimpanzees. These data revealed that humans and chimpanzees conserved the ancestral condition and that independent convergent pericentric inversion had occurred in the phylogenetic lines leading to gorilla and orangutan (Wienberg and others 1994) (Figure 3). In conclusion, the molecular cytogenetic data reveal that human chromosome 2 and its homologues in the great apes cannot be used to phylogenetically link human and chimpanzees to the exclusion of the gorilla.
Chromosome painting has also helped clarify exactly what additional interchromosomal rearrangements have occurred in human and great ape evolution. According to some cytogeneticists, there was a reciprocal translocation in the gorilla between homologues to human chromosome 5 and 17 (Dutrillaux 1975, Yunis and Prakash 1982). However, this conclusion was contested because gene mapping of the thymidine kinase locus (human chromosome 17) on the homologous great ape chromosomes apparently excluded this translocation in the gorilla (Seuànez 1987). Chromosome painting along with other more classical cytogenetic methods including high resolution Giemsa-banding, fluorochrome staining, and replication banding have since then all amply proven the existence of this translocation (Jauch and others 1992; Stanyon and others 1992; Wienberg and others 1990).
In situ hybridization did not reveal any other interchromosomal rearrangements asserted by cytogeneticists on the basis of chromosomal banding (Yunis and Prakash 1982). The painting of great ape chromosomes did confirm the similarity and dramatic conservation of syntenies between human and great ape karyotypes. However, in the lesser apes (hylobatids: gibbons and siamang), chromosomal painting revealed that chromosomal synteny was highly disturbed.
Lesser Apes
It has become generally appreciated that FISH greatly improves the utility of cytogenetic data for taxonomy and evolutionary studies. This was most clearly shown in studies on hylobatid chromosomes. Hylobatids are classified with great apes and human in the same primate superfamily Hominoidea; yet with chromosomal banding, they revealed no karyological relationship with great apes, humans, or any other primate species. Diploid numbers differ greatly (2n=38, 44, 50, 52) between hylobatids, and very few chromosome homologies could be found even within the genus. As a result they were systematically omitted from chromosomal phylogenies based on banding studies. The limited gene mapping studies restricted to 1 species (Hylobates concolor) were also not very helpful in elucidating chromosome homologies in these species (Cr6au-Goldberg 1993; Turleau and others 1983; Van Tuinen and Ledbetter 1989). Thus, it became clear that gibbons had experienced rapid and massive chromosome evolution and, as a consequence, had highly derived karyotypes (Marks 1983; Stanyon and Chiarelli 1983; Stanyon and others 1987; Van Tuinen and Ledbetter 1983). In contrast to the highly heterogeneous karyotypes, karyotypes of the hylobatids are fairly homogeneous in most other biological characteristics, revealing also that they are very closely related to great apes and humans. These results further confirm recent understandings (see Ferguson-Smith and others 1998) that dramatic changes in karyotypes do not necessarily imply dramatic changes in morphology. Molecular studies place gibbon divergence from pongids and humans at 16 to 23 million yr ago (MYAI), whereas orangutans diverged from 12 to 16 MYA and human and African apes about 5 to 10 MYA (Sibley and Ahlquist 1987).
All 4 hylobatid karyotypes have recently been studied by chromosome painting with human probes (Jauch and others 1992; Koehler and others 1995a,b; F. Yang and others, Cambridge University, personal communication, 1998). The detailed painting analyses of entire gibbon karyotypes delineated numerous translocations (Figure 2). However, as for other mammals, Ohno's principle that the X chromosome has been highly conserved during mammalian evolution (Ohno 1967) also appears applicable to gibbons. The mapping positions of paints were recorded on previously Giemsa-banded or on banded chromosomes stained with 4'6-diamidino-2-phenylindole 2 HC1. This method allowed a detailed comparison of classical and molecular cytogenetic findings. At least 33 translocations have occurred in the evolution of the Siamang karyotype. The 24 autosomes are composed of 60 recognizable segments that reveal DNA homology to regions of the 22 human autosomes. Only 2 autosomes have not been involved in translocations. The other lesser ape karyomorphs are also highly rearranged. The 25 H. concolor autosomes are composed of 69 recognizable segments that reveal DNA homology to regions of the 22 human autosomes; only 1 autosome, homologous to human chromosome 21, has not been involved in translocations. In Figure 4 is an example of summarized results obtained on the concolor gibbon included in an idiogram (Koehler and others 1995b). In Hylobates lar, the 20 gibbon autosomes are composed of 52 segments homologous to human autosomal chromosomes (Jauch and others 1992), whereas the hoolock gibbon (Hylobates hoolock) autosomes are fragmented into 57 segments (F. Yang and others, personal communication, 1998).
Profound karyological differences also exist between lesser apes. For example, since the phylogenetic separation of H. lar and H. syndactylus, these 2 hylobatids have independently accumulated numerous translocations (about 16 in HSY and 14 in HLA) in a relatively brief time. Thus, the 2 hylobatids are separated from each other by a total of 30 translocations and numerous intrachromosomal rearrangements. It is no wonder that chromosomal homologies even between these 2 hylobatids are very difficult to establish from banding patterns.
Chromosome painting also provides a new level of precision in comparative genome analysis for eliminating errors of confounding convergence with homology. The presence of a single metacentric, nucleolar organizer region (NOR1)-bearing, "marked" chromosome with a similar banding pattern in some gibbons and all Old World monkeys left little doubt for many cytogeneticists that lesser apes should be phylogenetically linked with monkeys instead of being classified with great apes and humans. Chromosome painting clearly revealed that the NOR chromosomes in lesser apes and monkeys were not homologous, but instead evolved by convergence (Stanyon and others 1995a).
The highly fragmented and intricately rearranged lesser ape karyotypes illustrate the utility of subchromosomal probes and reciprocal painting for high-resolution mapping. Reciprocal painting not only gives direct confirmation of chromosome homologies in 2 independent experiments but also provides additional information about subregional homologies (Figure 5). Microdissection probes specific for both arms of human chromosome 2 and various large insert probes cloned as YACs as well as reciprocal painting have been used to detail the homologies of human chromosome 2 in 1 of the H. lar-group species (Hylobates klossi) (Arnold and others 1996). In this species, the human chromosome 2 homologue is divided into 5 segments on 4 different gibbon chromosomes. The probes not only defined a homology map with a resolution of about 10 Mbp, but also delineated a breakpoint by a YAC, which spans a translocation (Figure 6). A more complete phylogenetic analysis will be possible when reciprocal painting between all hylobatid species and more YAC mapping data are available. These data will allow the various ancestral and common derived chromosome forms in the 4 different gibbon karyotypes to be distinguished.
Strikingly, the YAC defining a breakpoint in H. lar also spans a breakpoint in a human chromosome rearrangement. This YAC clone is part of a contig specific for the critical region for holoprosencephaly (a developmental defect of the midline of the human forebrain and midface) on human chromosome 2p21 (Schell and others 1996). This intriguing result suggests that the molecular tools may now exist to test the hypothesis that chromosome rearrangements are not random and that chromosome evolution and disease are correlated.
Gibbons are also unique among higher primates in revealing extensive chromosome polymorphisms that include not only inversions but also translocations (Couturier and Lernould 1991; Couturier and others 1982; Stanyon and Chiarelli 1983; Stanyon and others 1987; van Tuinen and Ledbetter 1983;). However, polymorphisms for inversions can also be observed within and between subspecies of the orangutan (deBoer and Seuànez 1982). Even though very few gibbons have been karyotyped all species have chromosome polymorphisms, which suggests that the process of karyological transformation is still under way. Chromosome painting identified inversions, a transposition and a translocation polymorphism. Apparently, all analyzed H. lar-species group gibbons analyzed share an inversion polymorphism on chromosome 8 (Jauch and others 1992; Stanyon and others 1983), indicating that the polymorphism is not transient but has even survived speciation events. However, for some heterozygote rearrangements--especially for various translocations--it is not totally clear whether they arose from breeding animals from different populations or subspecies. This unresolved problem must be further analyzed by including larger sample sizes with preferentially free-ranging individuals of known geographic origin.
The reason that hylobatids are so chromosomally derived remains elusive. It is not clear whether gibbons have a higher chromosomal mutation rate or the mutations that occurred are simply more easily fixed, or both. Aspects of gibbon social structure and ecology may have favored the rapid fixation of chromosome mutations. Gibbons are the only monogamous catarrhine species with nuclear family units and an arboreal lifestyle. In contrast, the chromosomally conservative Papionini (see Old World Monkeys) live in large terrestrial groups with multiple males and multiple females. Population bottlenecks and inbreeding also have been proposed to explain the rapid fixation of such rearrangements; however, the extensive chromosome polymorphism which is even shared by different species would argue against drastic bottlenecks during the divergence of gibbons. Nevertheless, the results clearly demonstrate that a "molecular clock" would not hold for chromosome rearrangements since, as discussed in more detail below, even more distantly related species than gibbons (various Old World monkeys and even carnivores) have much more conserved karyotypes compared with human.
Old World Monkeys
Old World monkeys are divided into 2 families: Cercopithecidae (baboons, macaques, guenons, and so forth) and Colobidae (African and Asian leaf-eating monkeys). Even though comparative data are far from complete at the time of this writing, painting with human chromosome specific probes has established whole chromosomal homology in species from both of these taxonomic divisions. In contrast to lesser apes, Old World monkeys are chromosomally conservative, and most chromosomal syntenies have been maintained intact. Only a few translocations have taken place in the evolution of these species.
Papionini. The Papionini, which include the genera Papio, Macaca, and Cercocebus, all have very similar karyotypes: 2n=42. The Japanese macaque, M. fuscata, was the first monkey to be studied with chromosome painting (Wienberg and others 1992). Strikingly, all human chromosome syntenies except the homologue for human chromosome 2 are found intact in the macaque karyotype. However, 3 macaque chromosomes were painted with 2 human chromosome-specific paints each (paints for chromosomes 7/21, 14/15, and 20/22), indicating entire chromosome fusion/fission events. Chromosome banding comparisons suggest that differences between species are limited to very few inversions. Therefore, it is likely that chromosome painting results in the Japanese macaque can be safely extrapolated to the entire group of more than 30 species (Stanyon and others 1988; Wienberg and others 1992). An important result of the chromosome painting revealed that chromosomal banding had correctly assigned only 80% of the chromosome homologies between macaques and humans (Wienberg and others 1992) although the karyotype is very similar to human. Yet it had been previously asserted, solely on the basis of banding patterns, that it was possible to trace the phylogeny of chromosomes in even more complex karyotypes from prosimians to man (Dutrillaux 1979, Dutrillaux and others 1986a,b).
Leaf-eating monkeys. With the exception of Nasalis larvatus (2n=48), the proboscus monkey, Colobidae species all have an identical diploid number: 2n=44. With classical staining, the only difference noted between African and Asian colobines was the presence of a small pair of acrocentric chromosomes in Asian species (Chiarelli 1963; Ushima and others 1964). Some cytogeneticists even proposed a strict phylogenetic relationship between colobines and hylobatids (Chiarelli 1963). With classical staining, the gibbon (H. lar-species group) appeared to have a karyotype identical to African colobines: the same diploid and fundamental number (number of chromosome arms) and the presence of I pair of "marked chromosomes." With chromosome banding, numerous differences between all African and Asian colobines were noted, but it was impossible to determine the relationship between colobines and gibbons (see above and Dutrillaux and others 1986a; Muleris and others 1986).
In the white and black colobus (Colobus guereza) the 24 human paints provided 31 signals on the autosomes (haploid male chromosome set) (Bigoni and others 1997a). Hybridization signals from 4 human paints were fragmented and found hybridized in various combinations on individual colobine chromosomes. Reciprocal translocations were found between human chromosomes I and 10, I and 17, as well as 3 and 19. The alternating hybridization signals between human 3 and 19 on colobus chromosome 12 reveal that, in this case, a reciprocal translocation was followed by a pericentric inversion. As in Papionini, chromosomes homologous to human 14 and 15 were associated. However, chromosomes homologous to human 21 and 22, which in Papionini are found in association with chromosomes 7 and 20, respectively, form the NOR-bearing marked chromosome in colobus.
Comparisons with hybridization patterns in other primates reveal that some Asian colobines have more derived karyotypes compared with African colobines, macaques, great apes, and humans. Published chromosome painting data are available for only 1 species of Asian colobines, the silvered leaf monkey (Presbytis cristata). In this species, the 24 human paints provided 30 signals on the haploid female chromosome set and 34 signals on the haploid male chromosome set. The difference between males and females is due to a reciprocal translocation between the Y and an autosome homologous to human chromosome 5. Translocations between the Y chromosome and an autosome are rare in primates. In catarrhines (Old World primates), P. cristata is the only species known to have a Y/autosome translocation and has produced a X1X2Y1Y2/X1X1X2X2 sex-chromosome system. Of 22 human autosomes, 18 are found intact in P. cristata. As in African colobines, homologues to human 14/15 and 21/22 formed associations. Multiple FISH signals provided by paint combinations of human chromosomes 1/19 and 6/ 16, respectively, indicate that these chromosomes have been split by reciprocal translocations. The signals of paint I and 19 also indicate additional intrachromosomal rearrangements on silvered leaf monkey chromosome 8 (Bigoni and others 1997b).
Chromosome painting also reveals for the douc langur (Pygathrix nemaeus) that chromosomal synteny is amply conserved compared with that of humans (Bigoni 1996). The 22 human autosomal paints provided 25 signals in the haploid karyotype. Fifteen human DNA paints each hybridized to a single P. nemaeus autosome. The chromosomes homologous to human 14/15 formed the same association as in other Old World monkeys, and the 21/22 association was the same as in the other colobines. One reciprocal translocation was present, involving the segments homologous to human chromosomes 1 and 19, which were found associated on 2 douc langur chromosomes. The hybridization pattern suggests that this may be the more ancestral form compared with that of the silvered leaf monkey since there is I less reciprocal translocation and apparently fewer intrachromosomal rearrangements.
New World Monkeys
Molecular cytogenetics could be particularly useful in the study of New World monkeys (Platyrrhini) and can contribute to the understanding of taxonomic relationships, which are still not well known. Various reports on New World monkeys have shown that these primates are much more karyologically variable than expected (Armada and others 1987; Clemente and others 1987; Ma 1981; Stanyon and others 1995b). Recent molecular cytogenetic data have shown that some taxa such as Atelidae (the genera Lagothrix, Brachyteles, Alouatta, and Ateles) are karyologically highly derived. The cytogenetic data also suggest that the number of species of New World monkeys is probably underestimated by researchers relying solely on traditional morphological data (Consigliere and others 1996; Morescalchi and others 1997). Although New World Primates have been divided into 2 or 3 families, the tendency is to divide them into the following 2: Callitrichidae and Cebidae. The monophyly of the Callitrichidae appears certain, but that of the Cebidae has been questioned frequently (Groves 1989). The chromosome data already available allow us to formulate hypotheses on some aspects of the phylogeny and taxonomy of New World monkeys.
Callitrichidae. Classical cytogenetic studies have shown that the Callitrichidae are relatively uniform cyto-genetically, with the diploid number from 2n=44 to 48. Banding analysis suggests that differences between species are due to only a few rearrangements (Canavez and others 1996; Nagamachi and others 1994). Molecular cytogenetic data on this taxon are limited to Callithrix jacchus (2n=46). The 24 human chromosome paints provided 33 signals per haploid male (Sherlock and others 1996). The synteny of 7 human homologous chromosomes (1, 2, 3, 8, 10, 13, and 15) is disturbed. There are 9 syntenic associations (multiple hybridization signals on single chromosomes) not found in humans.
Cebidae. Cebus capucinus. Chromosome painting in the capuchin monkey (C. capucinus; 2n=54) provided 34 signals in the haploid male karyotype (Richard and others 1996). Multiple hybridization signals on different chromosomes were seen for 8 human paints, and there are 6 chromosomal associations that are not found in humans.
Ateles geoffroyi. In the haploid karyotype of the black-handed spider monkey (Ateles geoffroyi; 2n=34), the total number of signals was 51 for the 22 human autosomal probes utilized, which is close to the highly rearranged gibbon karyotypes. Synteny was maintained on 9 homologous autosomes, but 6 of these were translocated to subregions of different spider monkey chromosomes. The other 13 human autosomal paints provided dispersed signals over the karyotype and provided multiple signals. There are 25 chromosomal associations not found in humans (Morescalchi and others 1997).
Alouatta. The howler monkeys are the best studied New World monkey genus, with publications on 3 species: Alouatta belzebul (2n=49, 50), Alouatta sara (2n=50), and Alouatta seniculus (2n=43, 44, 45). For howler monkeys, chromosome painting has revealed that the human chromosome homologues are often fragmented and translocated. It is clear that an exceptionally large number of chromosome rearrangements also separate the karyotypes of these 3 howler monkeys (Stanyon and others 1995b; Consigliere and others 1996, 1998). In A. belzebul, the total number of hybridization signals was 40. Paints from 13 human autosomes revealed conserved synteny, whereas 9 human chromosome paints each hybridized a howler chromosome along with some other human paints. The remaining 9 autosomal probes each provided multiple signals on a number of different howler chromosomes, revealing that they have been fragmented. There are 12 chromosome associations not present in humans. In A. sara, 44 hybridization signals were seen, and 10 homologous human chromosomes are fragmented in this karyotype. There are 16 chromosome associations different from those in humans. In A. seniculus, 45 signals were seen and 11 human homologous chromosomes are fragmented in this karyotype. There are 17 chromosome associations different from those in humans.
Comparisons among New World Monkeys
Several chromosome rearrangements observed in New World monkeys provide information about phylogeny. The association of segments homologous to human chromosome 8/18 material has been found in most New World monkeys studied so far and probably represents a derived rearrangement linking all Platyrrhini. The association 10/16 is apparently found in most Cebidae including Alouatta species, A. geoffroyi, and C. capucinus. The associations 5/7 and 15/22 are also found in A. geoffroyi, and the 4/15 and 5/1 I associations are known to occur in 2 Alouatta species. The association between regions homologous to human chromosomes 10/16 is an apomorphic ancestral condition for the Cebidae because it is found in all Cebidae studied, but not in C. jacchus. The syntenic association 5/7 and 2/16 could also be a common Cebidae apomorphism that was lost in howler monkeys. The syntenic associations 3/15 and 4/15 are probably apomorphic ancestral conditions for the subfamily Atelinae because they are present in both spider and howler monkeys. Other associations likely represent apomorphic rearrangements or convergences because they are not widespread among primates or among other mammals. It appears from chromosome painting data that New World monkeys are monophyletic. However, to determine the ancestral syntenic map for all branches of New World monkeys, additional studies are needed at the time of this writing.
Prosimians. The prosimians in general are usually assumed to be more primitive and therefore more similar to the ancestor of all primates than monkeys, apes, and humans. Indeed, some authors have suggested on the basis of chromosome banding that the chromosomes of some prosimian species are close to the ancestral primate karyotype (Dutrillaux 1979; Dutrillaux and others 1986a). Reciprocal chromosomal painting with human and lemur probes has recently provided a molecular cytogenetic definition and clarified the karyological evolution of 2 lemurs: Eulemur macaco macaco and Eulemur fulvus mayottensis (Müller and others 1997a). The results are particularly noteworthy because E.f. mayottensis has a banded karyotype very close to Microcebus murinus, the lemur species believed to have maintained (almost intact) the ancestral primate karyotype (Dutrillaux 1979; Rumpler and Dutrillaux 1976; Rumpler and others 1989).
Results indicate that the genome of both species has undergone only a few reciprocal translocations. However, additional translocations have occurred that make the karyotype even more different from humans than that of various nonprimate outgroup species (see below). Therefore, the suggestion that lemurs have maintained the ancestral primate karyotype cannot be confirmed. The ancestral primate karyotype must be based on chromosome forms symplesiomorphic for primates and outgroup taxa.
The synteny of about half of the human karyotype was conserved in the 2 lemurs. Reciprocal painting reveals that chromosomes homologous to human chromosomes 2q, 3, 9, 11, 13, 14, 17, 18, 20, and 21 are found entirely conserved in E. f mayottensis (that is, human chromosome paints provided only I pair of signals each). Although the synteny of genes on these chromosomes can probably be considered to be ancestral for all or most prosimians and higher primates in general, only 6 of these hybridized to entire lemur chromosomes (individual chromosomes homologous to human 2q, 11, 13, 17, 18, and 20). All other chromosomes formed additional associations with entire homologous chromosomes or fragments. Only a few more chromosome forms are also found in outgroup mammals (Table 1). Other painting experiments have also identified homologies between humans and other lemur species using some selected human chromosome probes (Apiou and others 1996).
The polarity of changes between the 2 lemur species can be determined by comparing the hybridization patterns found in other primates and various nonprimate mammals that function as outgroups (see below). E.f. mayottensis maintained 6 independent ancestral syntenic chromosomes, whereas E. m. macaco has conserved none. E. f mayottensis has 6 derived syntenic associations, whereas E. m. macaco has 15 derived syntenic associations. Clearly, E. m. macaco has a more derived karyotype than that of E. f mayottensis and has evolved by Robertsonian fusions from a karyotype similar to that of E. f mayottensis.
The other main taxonomic division in the prosimians is the Lorisidae. Studies with banding suggest that Otolemur crassicaudatus maintained a primitive karyotype ancestral for all Lorisidae and therefore is probably close to the proposed ancestral primate karyotype (Rumpler and others 1983). At the time of this writing, no literature has appeared on chromosome painting in Lorisidae; however, our unpublished data on O. crassicaudatus and Galago moholi reveal that 7 associations of different probes, or chromosomal syntenic groups, different from those of humans, are present in O. crassicaudatus while 17 are present in G. moholi. Among the 7 associations found in O. crassicaudatus, only 1 (14/15) is clearly ancestral for primates; the other 6 are apomorphic with respect to the ancestral condition for primates as indicated from the analysis of outgroup mammals (see below). The G. moholi karyotype reveals 15 associations that are apomorphic with respect to an ancestral primate condition. Five associations shared between the 2 loris taxa may be considered as candidates for the ancestral karyotype of the Lorisidae. However, before any proper hypothesis can be made about this group, it is necessary to gather data on other species of Lorisidae.
Contrary to the conclusions of the chromosome banding studies, the number of apomorphic chromosome syntenies found in both the Lemuridae and Lorisidae reveal that these species are not very close to the assumed ancestral primate karyotype. In addition to the 6 entirely conserved chromosomes, there are 9 syntenic associations in E.f. mayottensis of which only 3 can be considered ancestral for primates; namely chromosome painted by probes 3/21, 12/22, 14/15. O. crassicaudatus, which also has 6 apomorphic associations, is surprisingly equidistant from the syntenic condition of the probable ancestral primate karyotype. This conclusion, however, must be further confirmed by the analysis of more outgroup mammals.
CONCLUSION
From the primate chromosome painting data available at the time of this writing, and from the analysis of various nonprimate outgroup species, we can draw some conclusions about the ancestral primate karyotype. Nonprimate mammals have been studied by chromosome painting (Scherthan and others 1994) including carnivores (cat: Rettenberger and others 1995a; Wienberg and others 1997; O'Brien and others 1997; mink: Hameister and others 1997; harbor seal: Frönicke and others 1997) and ungulates (cow: Chowdhary and others 1996; Hayes 1995; Solinas-Toldo and others 1995; pig: Fr6nicke and others 1996; Goureau and others 1996; Rettenberger and others 1995b; sheep: Burkin and others 1997; deer species: Frönicke and Scherthan 1997; Yang and others 1997; horse: Raudsepp and others 1996).
Carnivores have karyotypes very similar to primates. Surprisingly, carnivores have karyotypes that are more similar to Old World monkeys, great apes, and humans than to prosimians and New World monkeys (see Table 1). The conservation of syntenies that correspond to entire human chromosomes is particularly informative. A recent review indicates that a majority (15) of human autosomes 3, 4, 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18, 20, and 21 were probably also syntenic in the outgroup and therefore also for the ancestral primate karyotype (Wienberg and Stanyon 1997). At least 10 were probably whole independent chromosomes (5, 6, 7, 9, 10, 11, 13, 17, 18, and 20), and others formed syntenic associations with other homologous human chromosomes.
Syntenic association of human homologous chromosomes or segments present in the karyotype ancestral to all primates certainly included the associations of segments homologous to human 3/21 and 14/15, 2 forms of chromosome 12/22 associations (chromosome 12/22a and 12/22b), and perhaps the association of homologous material to human chromosomes 4/8 and 16/19. Furthermore, some chromosomes were clearly fragmented in the ancestral primate karyotype, including homologous human chromosomes 1 and 2, which were each fragmented into 2 independent chromosomes. Chromosome 1 p and 2q form a large metacentric in carnivores, and gene mapping data reveal that a synteny for these chromosomes is also present in the mouse (Copeland and others 1993). In almost all primates, the 2q homologue is a separate chromosome, suggesting independent lq and 2q homologous in the ancestral primate kary-type. Other chromosomes split into 2 segments may include 4, 8, 16, and 19. Thus, a working hypothesis for the ancestral karyotype for all primates would contain the following homologous human chromosomes: 1a, 1b, 2a, 2b, 3/21,5, 6, 7, 9, 10, 11, 12/22a, 12/22b, 13, 14/15, 17, 18, 20, and X; also perhaps chromosomes 4/8, 8, 16, 16/19, 19, and Y. This hypothetical karyotype would have had a diploid chromosome number of 2n=48.
Firm conclusions about the origin of platyrrhine karyotypes require additional painting studies. However, chromosomal painting in rearranged karyotypes in New World monkeys promises to make a noteworthy contribution to species definition and conservation. The preliminary chromosome painting results in New World monkeys indicate that the homologies proposed on the basis of banding between New World primates and other primates contain many errors. Nevertheless, data from more species are needed to reconstruct the ancestral karyotypes for each branching point in platyrrhine evolution.
A few hypotheses can now also be made about the evolution of the karyotype ancestral to all living catarrhines (Old World primates including human). This evolution would have involved the fortnation of all syntenies present in humans with the exception of chromosome 2. The chromosome 14/15 association found in all outgroup mammals, pro-simians, and New World monkeys is also present in the ancestral catarrhine karyotype, and hominoids reveal the derived form of the single human chromosome 14 and 15 homologues. Two chromosomes present in the ancestral primate karyotype fused to make chromosome 1; a reciprocal translocation between 12/22a, and 12/22b would have formed homologues to 22 and 12. Fissions would have split the homologue to human 3/21 and, if present, those of the 4/8 and 16/19 associations found in outgroup mammals and prosimians, respectively. Additional translocations would have formed chromosomes 16 and 19. The diploid number of the ancestral catarrhine karyotype was probably 2n--46, including human homologous chromosomes 1, 2a, 2b, 3 to 13, 14/15, 16 to 22, X, and Y.
We caution that to firmly establish the ancestral karyotype for all primates hypothesized here, additional chromosome painting data on primates (especially both New World monkeys and prosimians) and selected outgroups are needed. Furthermore, most of the chromosome painting data available only allows the mapping of interchromosomal rearrangements (translocations). Intrachromosomal rearrangements such as inversions, transpositions, and centromere shills are also an important aspect of genome evolution, but they can be mapped using only subregional probes. Fine mapping using FISH (see Figure 6) should allow in the near future the mapping of breakpoints involved in the evolution of the primate genome and the relationship between chromosome change in evolution and disease. Finally, it is amply clear that the whole scheme of chromosomal phylogeny in primates should be restudied with the aid of molecular cytogenetics. The results could also be used to test assumptions about the role of chromosome changes in the evolutionary process and speciation.
1Abbreviations used in this paper: FISH, fluorescence in situ hybridization; MYA. million years ago; NOR, nucleolar organizer region; YAC, yeast artificial chromosome.
ACKNOWLEDGMENTS
We thank P. C. M. O'Brien for critical reading of the manuscript. This work was supported by grants from the Medical Research Council, the Deutsche Forschungsgemeinschaft, the Italian Ministry for Universities and Scientific Research.
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TABLE 1 Various ancestral priate homologous chromosomal syntenies observed in human, lemur, and nonprimate outgroup speciesa based on chromosome painting results.
| Ancestral chromosomes homologous to human (HSA) | Conserved in lemurs (EFM) | Conserved in cat (FCA) | Conserved in cattle (BTA) | Conserved in pig (SSC) |
| HSA 4 | - | - | SSC 8 | |
| HSA 6 | FCA B2 | - | - | |
| HSA 9 | FCA D4 | - | SSC 15 | |
| HSA 10 | FCA D2 | - | - | |
| HSA 11 | EFM 11 | FCA D1 | - | - |
| HSA 13 | EFM 9 | - | BTA 12 | SSC 11 |
| HSA 17 | EFM 15 | FCA E1 | BTA 19 | SSC 12 |
| HSA 18 | EFM 16 | - | BTA 24 | - |
| HSA 20 | EFM 17 | - | SSC 17 | |
| HSA X | EFM X | FCA X | BTA X | SSC X |
| - | - | - | - | |
| HSA 1p | EFM 2 | FCA F1 | BTA 3 | - |
| HSA 2qb | EFM8 | - | - | SSC 15 |
| HSA 3/21 | FCA C2 | BTA 1 | SSC 13 | |
| HSA 12/22 | EFM10, 19 | FCA B4 | BTA 5 | SSC 5 |
| HSA 14/15b | FCA B3 | - | - | |
| HSA 16q(?) | EFM 20 | FCA E3 | ||
| HSA 16/19 | FCA E2 | BTA 18 | - |
bChromosomes homologous to human chromosome 2p and to the association of chromosomes 14 and 15 are found conserved in many primates (Bigoni and others 1997a,b; Jauch and others 1992; Wienberg and others 1992), in the artiodactyls or the cat. These chromosomes are therefore considered to be ancestral for primates and carnivores.
FIGURE 1 Flow-sorted chromosomes provide the DNA probes for chromosome painting. Shown are 3 different chromosome-specific painting probes derived from the orangutan and hybridized to human chromosomes. The synteny of DNA in all 3 chromosomes is conserved, as it is in other chromosomes in both species except for the human chromosome 2, which has 2 homologues in primates. Image processing is used to produce false colors when merging the black and white images in a red/green/blue (RGB) format. (a) Orangutan paint labeled with cyanin-3-dUTP (red emission) denotes human chromosome 5. (b) Orangutan paint labeled with biotin-dUTP and visualized with avidin coupled to fluorescein (green emission) denotes human chromosome 8. (c) Orangutan paint labeled with digoxigenin-dUTP and visualized with an antidigoxigenin antibody coupled to cyanin-5 (infrared emission) denotes human chromosome 6. (d) Merging of the 3 images in red, green, and blue false colors.
FIGURE 2 Multicolor differentiation of human and primate chromosomes by chromosome painting. (a) Metaphase chromosomes and karyogram (b) of human chromosomes hybridized with all 24 different human chromosome specific paints and analyzed by spectral karyotyping. (c) Human chromosomes hybridized with gibbon (Hylobates syndactylus) paints labeled with 3 different haptens (see Figure 1). Gibbon chromosomes are highly rearranged, resulting in multiple signals on various human chromosomes. (d and e) Spectral karyotyping using human paints to chromosomes of the concolor gibbon (H. concolor), (d) metaphase plate, and (e) karyogram. Note that this animal is homozygous for the polymorphic chromosome forms 1b, 7b, and 22b (Figure 4). (f) Chromosome bar coding of human chromosomes. Subregional probes labeled in different colors are pooled to mimic a banding pattern after hybridization to chromosomes, a, b, and d are from Schr6ck and others 1996 (with permission from Science); e is from a collaboration with E. Schr6ck and T. Ried, National Institutes of Health, Bethesda, Maryland; and c and f are adapted from Müller and others 1997b and 1998, respectively.
FIGURE 3 Schematic summary of the hybridization patterns after fluorescence in situ hybridization with a chromosome 2q-specific paint obtained by microdissection of the entire long arm of human chromosome 2. The paint was hybridized to great ape and Old World monkey chromosomes. The human chromosome 2 idiogram (left) and the schematic drawing of the hybridization patterns are shown for human (HSA) and primate homologues (Pan troglodytes [PTR], Gorilla gorilla [GGO], Pongo pygmaeus [PPY], Macaca fuscata [MFU], and Cercopithecus aethiops [CAE]), without indicating the chromosome banding (adapted from Arnold and others 1995). The painted chromosome regions are indicated by a hatched pattern. Telomeric centromere (C-) banding in PTR, PPA, GGO, and CAE, as indicated by staining with 4'6-diamidino-2-phenylindole 2 HC1, is indicated by black bands. Also included are homologous hybridization patterns on human and great ape obtained by cosmids derived from the immunoglobulin V-kappa genes and related sequences (orphons), which map on both sides of the chromosome 2 centromere (Wienberg and others 1994). With respect to the position of the centromere, the hybridization patterns on the human chromosome 2p homologues in the chimpanzee are more similar to the patterns found in the outgroup (MFU and CAE) than those in the gorilla or the orangutan; in these species, the signals are found in the respective long arm. Therefore, convergent pericentric inversions probably occurred in different phylogenetic lines.
FIGURE 4 Idiogram of the karyotype of Hylobates concolor with the location of the hybridization signals of painting probes from single human chromosomes. The concolor chromosomes are numbered below and the human chromosome probes, to the right of the homologous concolor chromosome segments. Note a chromosome polymorphism that includes a translocation for chromosomes 1 and 22 (chromosome forms 1,1a, 22a, and 22b) and a pericentric inversion for chromosome 7 (7a and 7b) (reprinted from Koehler 1995b with permission from Genomics).
FIGURE 5 Schematic representation of reciprocal painting providing additional data on subregional homology between 2 species. In forward painting, a whole chromosome paint from species I paints homologous segments in 4 chromosomes of species 2, but the subregional origin of each segment is unknown. When painting probes from species 2 are then used in reverse painting, precise subregional assignments of each homologous segment can be made (after Müller and others 1997a).
FIGURE 6 Subregional mapping of complex chromosome rearrangements: hybridization patterns obtained with a painting probe obtained from human chromosome 2q, reciprocal painting with gibbon paints, and human yeast artificial chromosome clones (YACs), on human chromosome 2 and gibbon (Hylobates lar group species, HKL) homologous chromosomes. Extreme left, idiogram of human chromosome 2 (HSA 2) with the YAC locations. Right of HSA 2, reciprocal Hylobates lar gibbon (HLA)-specific chromosome paints mapped to HSA 2. Left of each gibbon, (HKL) chromosome, YAC locations. The underlined YAC 958d2 hybridizes on both HKL 10 and 16 and defines a translocation breakpoint. Right of HKL, chromosomes 1, 12, and 16, locations of the 2q microlibrary (2qML) hybridization signals (after Arnold and others 1996).
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