Comparative Mapping Using Chromosome
Sorting and Painting
Malcolm A. Ferguson-Smith, Fengtang Yang, and Patricia C. M. O'Brien
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 chromosomespecific (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
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.
'Abbreviations used in this article: DOP, degenerate oligonucleotideprimed; FACS, fluorescence-activated cell sorter; FISH, fluorescence in
situ hybridization; PCR, polymerase chain reaction.
68
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
colchicinized 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
ILAR Journal
the advent of the degenerate oligonucleotide-primed PCR
(DOP'-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 (Schrock 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 2000 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 basepair 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-voltage 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
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muntjac, 2n=7. (H) Male Chinese hamster, 2n=22. Axes reflect increasing fluorescence.
Volume 39, Numbers 2 and 3 1998
69
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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 lc), 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 Id)
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 for 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 brush-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
Volume 39, Numbers 2 and 3
1998
primate chromosome paints in human cytogenetic analysis
(Mtiller 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 (Miiller 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.
71
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
TABLE 2
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
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 (Consigned 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 autosomai paint probes hybridize
effectively to other autosomes, and it is usually easy to count
the number of separate blocks of homology per autosomai
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 autosomai
Estimates of number of conserved blocks of autosomai synteny between human and other species
Species
Autosomai
haploid blocks
Pan troglodytes
Presbytis cristata
Callithrix jacchus
Mustela vison
Felis catus
23
21
22
14
18
23
30
31
32
32
Aluatta seniculus sara
Equus caballus
Ovis aries
Sus scrofa
Muntiacus muntjac vaginalis
Bos taurus
Hylobates concolor
Mus musculus domesticus
21
31
26
18
3
29
25
19
41
42
42
46
48
56
63
116
72
TABLE 1 Ancient syntenies detected by chromosome
painting
Autosomai
no. conserved
Reference
Jauch and others 1992
Bigoni and others 1997
Sherlock and others 1995
Hameister and others 1997
Rettenberger and others 1995b, Wienberg and
others 1997
Consiglieri and others 1996
Raudsepp and others 1996
Burkin and others 1997a
Rettenberger and others 1995a
Yang and others 1997a
Solinas-Toldo and others 1995
Koehler and others 1995
Copeland and others 1993
ILAR Journal
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 (Miiller 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 (Muller
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 gouazoubird) have diploid numbers of 70. However, the Chinese muntjac (Muntiacus reevesi) has 46 chroVolume 39, Numbers 2 and 3
1998
mosomes, 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 be73
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75
13
14"
15
1
X+3
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.
come 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 nomogram. 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.
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.
ACKNOWLEDGMENTS
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
74
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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