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ILAR Journal V39(2/3) 1998
Comparative Gene Mapping
Malcolm A. Ferguson-Smith, Fengtang Yang, and Patricia C. M. O'Brien
| Malcolm A. Ferguson-Smith, M.B.,Ch.B., F.R.C.Path., F.R.C.P., F.R.S.E., F.R.S., is Professor and head of the Department of Pathology, Cambridge University, United Kingdom. Fengtang Yang, B.Sc., is a postgraduate student, and Patricia O'Brien, B.A., is a research technician in the same department. |
INTRODUCTION
The capacity to make whole chromosome-specific, fluorescence-labeled DNA probes, by random-primed polymerase chain reaction (PCR1) amplification of flow-sorted chromosomes or from chromosome-specific libraries, has led to a novel method of comparative genome mapping at the cytological level. This method has been developed using mammalian species. It has been found that when a chromosome-specific (paint) probe from 1 species is hybridized in situ to the chromosomes of another species, the paint probe detects large regions of homology containing genes conserved between the 2 species. Sometimes the probe hybridizes to only 1 chromosome, indicating that the whole chromosome is conserved. In other cases, several chromosomes or parts of chromosomes are painted, indicating that interchromosomal rearrangements have occurred during divergence of the 2 species. In general, more closely related species show fewer rearrangements than more distantly related species. Thus, only 1 interchromosomal rearrangement is found between the human and chimpanzee karyotypes, whereas at least 150 rearrangements are estimated between human and mouse (Nadeau and Taylor 1984). An important exception to this rule is the extensive rearrangement found between human and the lesser apes, greater even than that observed between humans and Old World monkeys.
Although regions of homology detected by chromosome painting may be regarded as similar to genetic linkage groups, they differ in that the order of genes within a region may be rearranged by inversions and other intrachromosomal changes detectable by linkage analysis but not by chromosome painting. These changes can be characterized by in situ hybridization using a series of conserved sequences cloned in yeast artificial chromosome or cosmid vectors, which may reveal differences in gene order between the species.
The homologies detected by comparative chromosome painting are gross phenomena; however, they are also useful for constructing simple maps of unmapped species, using chromosome paint probes from human or mouse species where the genetic maps are highly developed. Another use is in the study of karyotype evolution, either to determine the mechanisms of chromosomal changes that have occurred during evolution or to determine phylogenetic relationships. The method has much to offer in the study of the mammalian radiation.
METHODOLOGY
The technique of flow cytometry to sort chromosomes and to measure their DNA content was used first by Gray and others (1975) with fluid suspensions of chromosomes made from colchicinizcd cell cultures, stained with ethidium bromide and passed through the laser beam of a fluorescence-activated cell sorter (FACS1). Most of the chromosomes can be separated by size and sorted into their respective types without contamination with other chromosomes, and heteromorphisms and rearrangements have been found to be readily distinguished (Young and others 1981). The purity of each chromosome sort led to one of the first important applications-preparation of chromosome-specific libraries for constructing chromosome maps (Davies and others 1981 ).
Earlier FACS instruments used a single laser to generate fluorescence in chromosomes stained either by ethidium bromide or Hoechst 33258 (Sigma Chemical Company, Dorset, United Kingdom). Later, a dual laser system with Hoechst and chromomycin A3 staining was used for bivariate analysis in which the chromosomes were separated by both size and base-pair ratio. This technology provided better resolution and enabled additional chromosomes and even homologues to be separated. Chromosome libraries for all human chromosomes were soon available (Van Dilla and others 1986). Details of the current methodology for sorting chromosomes and for fluorescence in situ hybridization (FISH1) are given in Ferguson-Smith (1997).
The chromosome-specific libraries produced from flow sorting were found to be suitable in FISH experiments to paint individual chromosomes and to analyze chromosome rearrangements (Cremer and others 1988; Pinkel and others 1988). Improvements in resolution were achieved by reducing nonspecific background hybridization signals using the addition of unlabeled Cot-1 DNA to the probe to suppress repetitive, noncoding DNA (Lichter and others 1988). With the advent of the degenerate oligonucleotide-primed PCR (DOP1-PCR) technique for amplifying DNA, small numbers (300 to 500) of flow-sorted chromosomes could be used directly for the production of chromosome-specific paints (Telenius and others 1992), eliminating the need for libraries. Labeling was achieved by introducing into the PCR reaction nucleotides conjugated with various haptens (such as biotin or digoxigenin) or nucleotides directly labeled with fluorochromes (such as fluorescein isothiocyanate or Cy3); the former are detected by fluorochrome-conjugated streptavidin or fluorochrome-conjugated antibody, respectively. Chromosome preparations hybridized by the FISH technique are examined using digital fluorescence microscopy and image processing. Since several probes with different fluorochromes can be used together, a multicolor approach is possible. For example, the use of 24-color FISH and spectral karyotyping to discriminate between a complete set of human paint probes has made it possible to identify all interchromosomal rearrangements that have occurred during the divergence of human and the concolor gibbon in a single hybridization (Schröck and others 1996).
As an alternative to flow sorting, chromosome-specific probes can be made from microdissected chromosomes or parts of microdissected chromosomes (Meltzer and others 1992). This has a special application for comparative painting with avian and other species, which have multiple microchromosomes and are therefore difficult to sort, and for subregional studies of comparative homology.
FLOW KARYOTYPES
Chromosomes suspended in a polyamine buffer are analyzed in the flow cytometer at a rate of 200 to 200{) per sec. The fluorescence measurements from each chromosome are accumulated in large numbers in the FACS computer and used to construct a flow karyotype (Figure 1) composed of discrete clusters of signals, with each cluster representing 1 or more chromosome types arranged according to size and base-pair ratio. A-T rich chromosomes sort above a diagonal line drawn through the middle of the chromosome clusters whereas G-C rich chromosomes sort below this line.
After passage of the chromosome suspension through the 2 laser beams, the fluid stream is broken into a series of droplets, some of which contain a single chromosome. The fluorescence emitted from each chromosome is collected separately and stored in the FACS computer. Sorting is accomplished by applying an electrical charge to the droplets containing the chromosomes of interest so that they can be deflected into a container as they pass between 2 high-volt-age plates. The accumulation of signals can be observed on a video monitor and the chromosomes of interest selected for sorting by a simple gating procedure. Pure samples of 2 chromosome types can be collected simultaneously.
Flow karyotypes are characteristic for each individual and each species. Variation within a species is due largely to the presence of different amounts of noncoding repetitive DNA, present mostly as centromeric heterochromatin. In outbred species such as human, it is often possible to resolve chromosomes into their respective homologues because of the variation in amount of centromeric heterochromatin (Figure la). Similarly, the flow karyotypes of different inbred strains of the same species can often be distinguished because they are homozygous for different centromeric heteromorphisms. This has been well documented in the mouse (Ferguson-Smith 1997).
With some important exceptions, most closely related species tend to have similar flow karyotypes. Figure 1, a and b, compares the flow karyotypes of human and orangutan (Pongo pygmaeus). The main difference is the absence of the peak representing human chromosome 2 in the orangutan karyotype and the presence of 2 additional peaks (chromosomes 11 and 12, from which the human chromosome 2 is derived by fusion) in the orangutan karyotype. However, the flow karyotype of the white-cheeked gibbon (Hylobates concolor) (Figure 1c), which is one of the lesser apes, is quite different from the human and orangutan due to at least 31 different reciprocal translocations that have occurred during evolution from their common ancestor some 10 million years ago. In comparison, the flow karyotype of the more distantly related pig-tailed macaque (Macaca nemestrina) (Figure 1d) is more like that of the human as a result of fewer reciprocal translocations occurring during evolution from a common ancestor than observed in the lesser apes.
As an example of the variation in flow karyotypes observed between inbred strains, Figure 1, e and f, compares the PL/J strain of Mus musculus domesticus with the inbred Mus musculus castaneus. The variation in size is most marked tot chromosomes 1,7, 12, and 16 and the Y chromosome. These strain differences have been exploited in the production of a complete series of chromosome-specific paint probes for the mouse (Ferguson-Smith 1997). Species with low chromosome numbers and consequently larger chromosome size tend to show less variation in base-pair ratio. This generalization is illustrated for the Indian muntjac (Muntiacus muntjak vaginalis) with a diploid number of 2n=6 (in females) and 7 (in males) and the Chinese hamster (Cricetulus griseus) with 2n=22 (Figure 1, g and h), but it has also been observed in marsupial species including the tammar wallaby (Macropus eugenii, 2n=14) and the brash-tailed possum (Trichosurus vulpecula, 2n= 20).
CROSS-SPECIES CHROMOSOME PAINTING
Transcribed DNA tends to be conserved more or less unchanged across species. This has been shown extensively in mammalian species, but there are many examples of so-called housekeeping genes whose exons have been conserved more widely in the animal kingdom, including Drosophila melanogaster, Caenorhabditis elegans, and even in the yeast Saccharomyces pombe. Nontranscribed DNA, including most repetitive DNA, tends not to be conserved across species. The more rapid divergence of repetitive versus transcribed DNA has been exploited in the use of nonhuman primate chromosome paints in human cytogenetic analysis (Müller and others 1997a). Nonspecific background hybridization signals due to repetitive DNA are greatly reduced on human chromosomes, whereas chromosome-specific signals from transcribed DNA are retained. This phenomenon is responsible for the remarkable success of cross-species chromosome painting.
The 2 mammalian species with the most comprehensive genetic maps are the human and the mouse. Comparative mapping reveals that the mouse genome has been extensively rearranged during evolution, compared with the human genome. It is estimated that the 22 haploid autosomal syntenic groups in the human are rearranged into at least 116 separate groups in the mouse (Copeland and others 1993). These groups should correspond to regions of homology revealed by hybridizing human chromosome-specific painting probes to mouse chromosomes. Preliminary work from this laboratory with results now available from most chromosome comparisons (Ferguson-Smith 1997) indicates that this assumption is correct. Cross-species comparative painting can therefore be relied on to indicate regions of genetic map homology.
The earliest comparative FISH studies used chromosome-specific human DNA libraries as painting probes on the chromosomes of other primates (Jauch and others 1992; Koehler and others 1995; Wienberg and others 1990). These studies demonstrated the value of comparative chromosome painting in problems of cytotaxonomy and in studies of genome rearrangements that had occurred during evolution. Scherthan and others (1994), using several human chromosome-specific libraries, demonstrated homologous segments in chromosomes of mouse, muntjac, and the fin whale, thus extending the technique to a wide range of nonprimate mammals. Extensive comparative studies have now taken place in cattle (Solinas-Toldo and others 1995), pig (Rettenberger and others 1995a), and horse (Raudsepp and others 1996) using these libraries. Paint probes prepared from flow-sorted chromosomes have proved to be superior for cross-species hybridization, and human probes made available from this laboratory have been used in comparative studies in a number of species including marmoset, cattle, pig, and mink (Goureau and others 1996; Hameister and others 1997; Hayes 1995; Sherlock and others 1995). More recently, paint probes have been developed from sorted chromosomes of species other than human, including mouse (Rabbitts and others 1995), pig (Langford and others 1993; Milan and others 1996), cattle (Schmitz and others 1995), dog (Langford and others 1996), lemurs (Müller and others 1997b), Indian and Chinese muntjacs (Yang and others 1995, 1997a,b,c), brown brocket deer (Yang and others 1997d), tammar wallaby (Toder and others 1997), chicken (Ambady and others 1997), and sheep (Burkin and others 1997a).
Comparative studies confirm that cross-species painting is far more reliable than conventional cytogenetics using Giemsa banding in determining chromosomal homology between species. Several misleading conclusions from earlier Giemsa banding have been refuted by cross-species painting. For example, the conclusion that nucleolus-organizing chromosomes were shared between lesser apes and Old World monkeys was found to be incorrect (Stanyon and others 1995) as was the postulated translocation in the orangutan that was thought to be homologous to human chromosomes 8 and 20 (Jauch and others 1992).
Comparative studies of human homologies in the chromosomes of other, more distantly related mammalian orders (outgroups) help to define ancestral karyotypes. It is to be expected that blocks of chromosomal DNA that are syntenic in both human and 1 or more outgroup species are likely to be ancestral to them all. All primates studied so far have a number of chromosomes that are painted exclusively by 1 human chromosome-specific painting probe. The same is found for a smaller number of chromosomes in many outgroup species. For example, human chromosome 17 paint probe hybridizes to only 1 chromosome in almost all mammalian species studied to date, indicating that it represents 1 of the more ancient syntenic groups. Some rearrangements that have occurred during the evolution of humans and great apes have been revealed by chromosome painting with human probes. For example, homologues of human chromosomes 14 and 15 are separate in the great apes but linked in lower primates and nonprimate species. In addition, parts of different human chromosomes such as chromosomes 3 and 21 are linked in species as different as the tree shrew, cat, pig, and mouse (for example, Rettenberger and others 1995a,b; Wienberg and Stanyon 1995). Other examples are listed in Table 1 and are representative of more recent rearrangements that have occurred in the evolution of the great apes and humans. Similar types of association help to link other species within a particular branch of the phylogenetic tree. For example, among the New World monkeys, the marmoset and capuchin monkey share a translocation between human chromosomes 8 and 18 (Richard and others 1996; Sherlock and others 1996), and the different species of red howler monkey share translocations between human chromosomes 2 and 16 and 10 and 16 (Consiglieri and others 1996). The red howler monkey species are otherwise remarkable for their karyotypic variability.
Comparing the number of separate conserved blocks of chromosome synteny revealed in the chromosomes of different mammals by hybridizing with human chromosome-specific paint probes gives some indication of phylogenetic relationships. In all mammals, the human X-specific paint hybridizes to the X chromosome. As the Y chromosome has diverged more rapidly than the X by losing some DNA sequences and acquiring others, the human Y chromosome paint tends to hybridize to other Y chromosomes minimally, if at all. However, human autosomal paint probes hybridize effectively to other autosomes, and it is usually easy to count the number of separate blocks of homology per autosomal haploid set in each species. The results obtained for a number of species are shown in Table 2. The chimpanzee (Pan troglodytes), with 46 autosomes, has 23 blocks of autosomal homology. Apart from human chromosome 2, which resulted from fusion of the chimpanzee chromosomes 12 and 13, the number of blocks corresponds to the number of human autosomes, indicating that no other gross interchromosomal rearrangement separates humans from chimpanzees. Subregional painting, however, reveals that a number of inversions and possibly other intrachromosomal rearrangements have occurred in the divergence of humans and the great apes (Müller and others 1997c). It can be seen in Table 2 that fewer interchromosomal changes have occurred in the more distantly related cattle and sheep than among the more closely related gibbons. Clearly the rate of occurrence of interchromosomal rearrangements differs in different taxa and is not invariably related to evolutionary time, although the mouse, which is separated from humans by at least 85 million years, shows the greatest number of rearrangements encountered so far.
Since chromosomes can be sorted and made into chromosome-specific paints from any species for which cell cultures are available, the possibility of reciprocal cross-species chromosome painting is now being realized. When a chromosome paint from species A hybridizes to regions of 3 chromosomes in species B, it is obviously important for genetic mapping to determine which part of the species A chromosome maps to each of the 3 chromosomes from species B. This can be done by sorting the 3 chromosomes from species B and painting them back separately (or in different colors) to the species A chromosome. The orientation of each of these blocks cannot be determined by reciprocal painting, but even this question can be resolved by using locus-specific (conserved) gene probes, cloned in a suitable cosmid or bacterial artificial chromosome vector, to mark the ends of each syntenic block. When 2 or more separate blocks from 1 chromosome in species A map to the same chromosome in species B, the origin of each block can be determined either by using subregional probes derived by microdissection or by a series of locus-specific gene probes.
Reciprocal cross-species chromosome painting has been achieved and published in several species including muntjacs (Yang and others 1995, 1997b,c,d), pig (Goureau and others 1996; Milan and others 1996), cat (Rettenberger and others 1995b; Wienberg and others 1997), and lemur (Müller and others 1997b). In all cases in which gene mapping information is available, the chromosome painting evidence of homology is generally consistent.
KARYOTYPE EVOLUTION IN MUNTJAC SPECIES
Cross-species comparative chromosome painting has been used to investigate the remarkable variation in chromosome number and karyotype in deer species. High chromosome numbers are common; for example, both the reindeer (Rangifer tarandus caribou) and the brown brocket deer (Magama gouazoubira) have diploid numbers of 70. However, the Chinese muntjac (Muntiacus reevesi) has 46 chromosomes, and the Indian muntjac has 6 large chromosomes in the female and 7 in the male, representing the lowest chromosome number in mammals. Despite the difference in chromosome number, the phenotypes of the Chinese and Indian muntjacs are similar, and they breed to produce viable but sterile offspring.
The results of cross-species hybridization suggest that the ancestral chromosome number in deer species probably approximates 70 and that the reduction in chromosome number in the muntjacs is mainly the result of multiple chromosome fusions (Yang and others 1995, Yang and others 1997a,b,c,d). Thus, the Chinese muntjac karyotype can be reconstructed by 12 tandem fusions from the 70-chromosome karyotype of the brown brocket deer (Yang and others 1997d). All deer chromosomes share a class of repetitive centromeric DNA, detectable by hybridization with the C5 probe (Lin and others 1991), and remnants of this centromeric repeat can be found at many of the sites of tandem fusion. This observation is consistent with the view that most of the fusions involve whole chromosomes and are the result of centromere-telomere tandem fusions. It has also been noted that the few ancestral rearrangements present in the Chinese muntjac have breakpoints at the sites of tandem fusion. It appears likely that these are sites of preferential recombination during meiosis because they share a common DNA sequence. It might follow that the mechanism responsible for tandem fusion is recombination between homologous regions on nonhomologous chromosomes, as postulated for the origin of Robertsonian translocations in humans (Ferguson-Smith 1973).
A UNIVERSAL MAPPING NOMOGRAM
The Indian muntjac has attracted the attention of mammalian cytogeneticists since its large chromosomes and small chromosome number were first described by Wurster and Benirschke (1970). It proved appropriate for study by FISH as each chromosome could be separately painted in 1 of 3 colors (Yang and others 1995). In addition to human (Yang and others 1997a), a number of other species have had their chromosome-specific paint probes hybridized to Indian muntjac chromosomes. These include not only the deer species mentioned above, but also the sheep Ovis aries (Burkin and others 1997a,b). Alignment of hybridization results for these species against the Indian muntjac idiogram is shown in Figure 2. Homologies with cattle (Bos taurus) chromosomes have been assigned from FISH results with human probes (Hayes 1995; Solinas-Toldo and others 1995). The various chromosomal homologies between these species can be read easily from this preliminary nomogram, and when the studies are completed, it will be necessary only to add the regional limits (in terms of the nomenclature established in 1978 by the Standing Committee on Human Cytogenetic Nomenclature [SCHCN 1978] and commonly referred to as "ISCN"). It should be possible to add the results of comparative painting in any mammalian species as the data become available. The nomogram has value in directing gene mapping studies in unmapped species. It also provides information on the segregation of syntenic blocks during evolution. For example, chromosomal segments that have become separated during primate evolution but remain linked in other species (and are therefore ancestral) can be identified from the homogram. As noted by others (for example, Rettenberger and others 1995b), regions homologous to human chromosomes 3/21, 12/22, 14/15, and 16/19 are found linked on the same chromosome in each of the other species shown in Figure 2. In this diagram, asterisks mark the sites of hybridization of the C5 centromeric probe in the Indian muntjac. Each of the ancestral linkages is flanked by the C5 centromeric sequence indicative of the site of an ancient tandem fusion.
CONCLUSION
Chromosome-specific painting probes have been generated by DOP-PCR amplification of sorted chromosomes from many species. When used in cross-species FISH experiments, these probes recognize conserved DNA sequences and reveal that across all mammals studied, relatively large blocks of chromosomal DNA have been transmitted virtually unchanged throughout evolution. Genetic mapping data confirm that conserved genes within genetic linkage groups are retained in these blocks as expected from their chromosomal location in the extensively mapped genomes of mouse and human. It follows that the location of the equivalent linkage groups in unmapped species may be determined by reciprocal cross-species chromosome painting. A simple nomogram based on the idiogram of the Indian muntjac has been constructed using results from chromosome painting experiments in several species. This example illustrates the relative ease with which cross-species comparisons of the genomes of mammals can be made to guide the genetic mapping of unmapped species. In addition, cross-species chromosome painting has considerable potential in determining phylogenetic relationships of extant species in the mammalian radiation, as well as being a powerful tool for understanding the nature of chromosomal reorganization in evolution.
1Abbreviations used in this article: DOP. degenerate oligonucleotide-primed; FACS, fluorescence-activated cell sorter; FISH, fluorescence in situ hybridization; PCR, polymerase chain reaction.
ACKNOWLEDGMENTS
The work described in this paper was supported by a Medical Research Council Programme Grant (MAF-S). We especially thank Deborah Walsh for expert assistance with cell cultures.
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TABLE 1 Ancient syntenies detected by chromosome painting
| Human chromosome | Pattern conserved in |
| 1 and 2 | Cat, pig, cow, muntjac |
| 3 and 21 | Cat, pig, cow, muntjac |
| 14 and 15 | All mammals except apes |
| 16 and 19 | Cat, pig, cow, muntjac |
| 12 and 22 | All nonprimates |
TABLE 2 Estimates of number of conserved blocks of autosomal synteny between human and other species
| Species | Autosomal haploid blocks | Autosomal no. conserved | Reference |
| Pan troglodytes | 23 | 23 | Jauch and others 1992 |
| Presbytis cristata | 21 | 30 | Bigoni and others 1997 |
| Callithrix jacchus | 22 | 31 | Sherlock and others 1995 |
| Mustela vison | 14 | 32 | Hameister and others 1997 |
| Felis catus | 18 | 32 | Rettenberger and others 1995b, Wienberg and others 1997 |
| Aluatta seniculus sara | 21 | 41 | Consiglieri and others 1996 |
| Equus caballus | 31 | 42 | Raudsepp and others 1996 |
| Ovis aries | 26 | 42 | Burkin and others 1997a |
| Sus scrofa | 18 | 46 | Rettenberger and others 1995a |
| Muntiacus muntjac vaginalis | 3 | 48 | Yang and others 1997a |
| Bos taurus | 29 | 56 | Solinas-Toldo and others 1995 |
| Hylobates concolor | 25 | 63 | Koehler and others 1995 |
| Mus musculus domesticus | 19 | 116 | Copeland and others 1993 |
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FIGURE 2 Idiogram of the Indian muntjac female karyotype showing homologies revealed by chromosome painting for human (HSA), sheep (OOV), bovine (BTA), Chinese muntjac (MRE), and brown brocket deer (MGO). The idiogram acts as a nomogram to guide comparative mapping studies against the extensively mapped human genome. Asterisks show sites of hybridization of the muntjac C5 centromeric probe, indicating sites of ancestral fusion. Not all chromosomes from the brown brocket deer were available for painting.
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