The evolution of CMA bands in <Emphasis Type="Italic">Citrus</Emphasis> and related genera

Most species of Citrus and related genera display a similar karyotype with 2n = 18 and a variable number of terminal heterochromatic blocks positively stained with chromomycin A₃ (CMA⁺ bands). Some of these blocks are 45S rDNA sites, whereas others may correspond to the main GC-rich satellite DNA found in several Citrus species. In the present work, the distribution of the 45S rDNA and the main satellite DNA isolated from C. sinensis (CsSat) were investigated by in situ hybridization in seven species of Citrus, two species of closely related genera (Fortunella obovata and Poncirus trifoliata) and four species of the subfamily Aurantioideae, which were less related to Citrus (Atalantia monophylla, Murraya paniculata, Severinia buxifolia, and Triphasia trifolia). In Citrus, Fortunella, and Poncirus, most CMA⁺ bands colocalized only with CsSat sites, whereas others colocalized only with rDNA sites. However, some of these species displayed a few CMA⁺ bands that colocalized with sites of both probes and other CMA⁺ bands that did not colocalized with any of the probes. On the other hand, in the four species less related to Citrus, no CsSat signal was found on chromosomes. On Southern blot, the CsSat probe hybridized with genomic DNA from Citrus, Fortunella, and Poncirus at high stringency only, while under the less stringent conditions, it also hybridized with distantly related species. Therefore, CsSat sequences are the principal component of the heterochromatic blocks of Citrus, Poncirus, and Fortunella, whereas CsSat-like sequences seem to be widespread in the subfamily Aurantioideae. These data further suggest that the variable number of terminal CMA⁺ bands observed on chromosomes of Citrus and related genera are probably the consequence of amplification or reduction in the number of CsSat-like sequences distributed on chromosome termini, paralleled by mutation and homogenization events, as proposed by the library hypothesis.     

Complex rearrangements are involved in <Emphasis Type="Italic">Cephalanthera</Emphasis> (Orchidaceae) chromosome evolution

The genus Cephalanthera is an excellent plant group for karyotype evolution studies because it exhibits a dysploid series and bimodal karyotypes. With the aim of understanding their chromosomal and phylogenetic relationships, rRNA genes and the Arabidopsis-type telomeric sequence were mapped by fluorescence in-situ hybridization (FISH), and the rDNA intergenic spacer (ITS) was sequenced for the first time in three European species: C. longifolia (2n = 4x = 32), C. damasonium (2n = 4x = 36) and C. rubra (2n = 4x = 44). One 45S and three 5S rDNA sites are observed in C. longifolia, one 45S and two 5S sites in C. damasonium, and two 45S and one 5S site in C. rubra. Telomeric signals were observed at every chromosome end in all three species and C. damasonium also displays interstitial signals on three chromosome pairs. In agreement with chromosome data, molecular analyses support C. longifolia and C. damasonium as closely related taxa, while C. rubra stands apart. Possible pathways of karyotype evolution are discussed in reference to a previous hypothesis. The results indicate that complex chromosomal rearrangements, possibly involving Robertsonian fusions and fissions, loss of telomeric repeats, gain or loss of rDNA sites and other heterochromatic sequences and inversions, may have contributed to generating the present-day karyotypes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chromosomal study of a lamprey (<Emphasis Type="Italic">Lampetra zanandreai</Emphasis> Vladykov, 1955) (Petromyzonida: Petromyzontiformes): conventional and FISH analysis

Karyotype and other chromosomal characteristics in the Adriatic brook lamprey Lampetra zanandreai, representative of one of the most ancestral group of vertebrates, were examined using conventional (Ag-staining, C-banding as well as CMA₃ and DAPI fluorescence) and molecular (FISH with 18/28S rDNA and EcoRI satDNA as probes) protocols with metaphase chromosomes derived from whole blood cultures. The chromosome complement had a modal diploid chromosome number of 2n = 164, as in other petromyzontid lamprey species. Ag-staining and CMA₃ fluorescence, as well as FISH with 18/28S rDNA probes, detected nucleolar organizer regions (NORs) close to the centromeres of the biarmed chromosomes of pairs 1 and 2, the largest chromosome pairs of the complement. In addition to NORs, CMA₃ fluorescence revealed positive signals in approximately 40 other chromosomes. DAPI stained mostly centromeric regions of many chromosomes as well as conspicuously massive blocks overlapping NOR sites. C-banding evidenced a large amount of constitutive heterochromatin in somatic chromosomes, with approximately 40 C-positive acrocentric elements completely heterochromatic, corresponding with the 40 CMA₃+ chromosomes and positive heterochromatic blocks in pericentromeric regions of chromosome pairs 1 and 2. Polymerase chain reaction (PCR)-based cloning of satellite DNA with primers derived from Petromyzon marinus centromeric sequences was successful for L. zanandreai genomic DNA. The sequence was AT-rich (59%) and characterized by short consensus motifs similar to other centromeric satellite motifs. FISH using satDNA clones as a probe produced a fluorescent signal on a single pair of small chromosomes. This sequence was PCR-amplified also in L. planeri and P. marinus genomic DNA, and the evolution of this repetitive element in the above species was analysed.     

Genome size, karyotype, meiosis and a novel extra chromosome in Torenia fournieri, T. baillonii and their hybrid

Torenia is a suitable model plant to study plant fertilization because of its protruding embryo sac. However, information on the genomes and chromosomes of this species is limited. We determined the genome sizes of T. fournieri Linden and T. baillonii Godefr as 1.71 pg × 10⁸ bp and 1.67 × 10⁸ bp, respectively. The small genome size of these species suggests their superiority as the targets for molecular cloning studies. Furthermore, karyotypes of T. fournieri and T. baillonii were determined using FISH probed with 5S rDNA, 45S rDNA and species-specific centromere repetitive sequences. Although the two species have similar genome size, number of chromosomes, centromere repeats and 5S rDNA loci were varied. Observation of meiosis in the F₁ hybrid revealed that all chromosomes except one of T. fournieri paired well with the chromosomes of T. baillonii throughout the entire length of the chromosomes including species-specific centromeric regions. One exceptional chromosome of T. fournieri behaved as a univalent and was not always required for gametogenesis. The present results provide the basis for the molecular genetics in Torenia.     

Possible autosomal origin of macro B chromosomes in two grasshopper species

The acrocentric macro B chromosomes of Rhammatocerus brasiliensis (Acrididae, Gomphocerinae) and Xyleus discoideus angulatus (Romaleidae, Romaleinae) are highly similar to the X chromosome in each species in terms of morphology, size, and pycnosis. However, the results of FISH experiments using 45S and 5S rDNA probes suggest that in both species the B chromosomes are most likely of autosomal origin. In R. brasiliensis, the B chromosome presented 5S rDNA but not 45S rDNA, in resemblance to the L₂, L₃, M₅ and S₁₁ autosomes, but the X chromosome lacks both rDNA families. In X. d. angulatus, 45S rDNAs is absent from the B chromosome, whereas the X chromosome contains one of the two 45S rDNA clusters in the genome. The occurrence of B chromosomes in all nine R. brasiliensis populations analyzed indicates that they are widely distributed in Northeastern Brazil, and the small amount of interpopulation variation found for B chromosome prevalence suggests the existence of high gene flow, presumably due to the abundance of this grasshopper species on several types of vegetation and its relatively high flight capability.     

Chromosomal Characteristics of Ribosomal DNA in the Primitive Semionotiform Fish, Longnose Gar Lepisosteus osseus

The chromosomes of longnose gar, Lepisosteus osseus, an extant representative of early radiation of actinopterygian fishes, were studied using conventional Giemsa-staining, Ag-staining, CMA₃-fluorescence and fluorescence in-situ hybridization (FISH). The diploid chromosome number was 2n = 56 and the karyotype contained 11 pairs of metacentric, 6 pairs of submetacentric, 3 pairs of subtelocentric macrochromosomes and 16 microchromosomes. Nearly all macrochromosomes showed large CMA₃-positive regions resembling the R-bands of higher vertebrates, indicating extensive distribution of GC-rich DNA along chromosomes. The nucleolar organizer regions (NORs) were located on the end of the short arm of a single small metacentric macrochromosomal pair. These sites were strongly CMA₃-positive, suggesting that ribosomal sites are associated with GC- rich DNA. In-situ hybridization (FISH) with a rDNA probe gave consistently positive signals in the same regions detected by Ag- staining and CMA₃-fluorescence. The evolutionary conservation of positive CMA₃-fluorescence of ribosomal sites in holostean and teleostean fishes is discussed.     

Chromosome homeologies and high variation in heterochromatin distribution between <Emphasis Type="Italic">Citrus</Emphasis> L. and <Emphasis Type="Italic">Poncirus</Emphasis> Raf. as evidenced by comparative cytogenetic mapping

The genus Citrus is well-known for its economic importance and complex taxonomy. Only three to six Citrus taxa are considered true biological species, among them is Citrus medica, the citron. Previous studies based on chromomycin A3 (CMA)/4′,6-diamidino-2-phenylindole staining showed that the citron has a homomorphic karyotype, consisting of four distinct chromosome types according to the CMA⁺ heterochromatin distribution. Based on the pattern of CMA⁺ bands and the hybridization of 25 bacterial artificial chromosomes (BACs) from a genomic library of Poncirus trifoliata, a closely related species to the genus Citrus, we were able to identify each chromosome pair and build a comparative cytogenetic map for C. medica. The data showed a high degree of sequence conservation between these genera, enabling heterologous hybridization of BACs, and the establishment of chromosomal homeologies. It was thus possible to visualize changes in the position of some BACs in relation to CMA⁺ bands. Since no breakdown of synteny was observed between these species, expansions and contractions in repetitive DNA sequences seem to be the major driving force of chromosomal evolution since the separation of these two genera.     

Micromechanical studies of mitotic chromosomes

Mitotic chromosomes respond elastically to forces in the nanonewton range, a property important to transduction of stresses used as mechanical regulatory signals during cell division. In addition to being important biologically, chromosome elasticity can be used as a tool for investigating the folding of chromatin. This paper reviews experiments studying stretching and bending stiffness of mitotic chromosomes, plus experiments where changes in chromosome elasticity resulting from chemical and enzyme treatments were used to analyse connectivity of chromatin inside chromosomes. Experiments with nucleases indicate that non-DNA elements constraining mitotic chromatin must be isolated from one another, leading to the conclusion that mitotic chromosomes have a chromatin ‘network’ or ‘gel’ organization, with stretches of chromatin strung between ‘crosslinking’ points. The as-yet unresolved questions of the identities of the putative chromatin crosslinkers and their organization inside mitotic chromosomes are discussed.     

Major and 5S Ribosomal Sequences of the Largemouth Bass Micropterus Salmoides (Perciformes, Centrarchidae) are Localized in GC-Rich Regions of the Genome

Major and 5S ribosomal genes have been localized in the chromosomes of Micropterus salmoides. By C-banding, Ag-staining, CMA₃-staining and 45S and 5S fluorescence in-situ hybridization (FISH), we demonstrate that the 45S and 5S ribosomal genes are clustered in two different chromosome pairs and both are located in heterochromatic GC-rich regions. PCR amplification and sequencing of the 5S intergenic non-transcribed sequences have allowed us to identify variability essentially due to a trinucleotide tandem repeat (GCT). This revised version was published online in August 2006 with corrections to the Cover Date.     

Additional locus of rDNA sequence specific to the X chromosome of the liverwort, Marchantia polymorpha

The molecular cytogenetic organization of 17S ribosomal RNA genes (17S rDNA), a part of the 45S rDNA repeat, was investigated on the chromosomes of the liverwort Marchantia polymorpha using fluorescence in-situ hybridization (FISH). The numbers of 17S rDNA loci visualized in female and male chromosomes were ten and nine, respectively. This heterogeneous localization was due to the presence of an additional 17S rDNA locus on the X chromosome and its absence on the Y chromosome. The signal on the X chromosome covered almost the entire region of its long arm. The other nine signals were observed on the same loci of respective autosomes in both sexes. Southern hybridization analysis revealed an additional band including 17S rDNA exclusively on EcoRI digested female genomic DNA supporting the existence of an additional 17S rDNA locus on the X chromosome.     

Integration of cytogenetic and genetic linkage maps of <Emphasis Type="Italic">Lotus japonicus</Emphasis>, a model plant for legumes

To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.     

Ribosomal DNA locus evolution in Nemesia: transposition rather than structural rearrangement as the key mechanism?

We investigated chromosome evolution in Nemesia using fluorescent in-situ hybridization (FISH) to identify the locations of 5S and 45S (18–26S) ribosomal genes. Although there was conservation between Nemesia species in chromosome number, size and centromere position, there was large variation in both number and position of ribosomal genes in different Nemesia species (21 different arrangements of 45S and 5S rRNA genes were observed in the 29 Nemesia taxa studied). Nemesia species contained between one and three pairs of 5S arrays and between two and four pairs of 45S arrays. These were either sub-terminally or interstitially located and 45S and 5S arrays were often located on the same chromosome pair. Comparison of the positions of rDNA arrays with meiotic chromosome behaviour in interspecific hybrids of Nemesia suggests that some of the changes in the positions of rDNA have not affected the surrounding chromosome regions, indicating that rDNA has changed position by transposition. Chromosome evolution is frequently thought to occur via structural rearrangements such as inversions and translocations. We suggest that, in Nemesia, transposition of rDNA genes may be equally if not more important in chromosome evolution.     

Classical and molecular cytogenetics of <Emphasis Type="Italic">Khawia sinensis</Emphasis> (Cestoda: Caryophyllidea), invasive parasite of carp, <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

Chromosomes of the invasive tapeworm Khawia sinensis (Caryophyllidea), the specific parasite of common carp, were analyzed by means of conventional Giemsa staining and using fluorescent DAPI and YOYO-1 dyes, silver staining, and fluorescent in situ hybridization (FISH) with 18S rDNA probe. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n = 3m + 5t, TCL = 42.54 μm). Constitutive heterochromatin was located at pericentromeric regions of all pairs, except for the largest metacentric pair (no. 1), which possessed no DAPI-positive band. FISH with rDNA probe revealed that both homologues of chromosome pair no. 6 carry a cluster of ribosomal arrays, which were located interstitially close to the centromere. Present results are compared with previous cytogenetic data on Khawia spp., and comments are made on the karyotypes with respect to their phylogenetic links.     

Exclusive Localization of Tandem Repetitive Sequences in Subtelomeric Heterochromatin Regions of Leymus Racemosus (Poaceae, Triticeae)

Two kinds of tandem repetitive sequences were isolated from Leymus racemosus (Lam.) Tzvelev. One of them was classified in the 350-bp family originally isolated from Secale. The other was a novel repetitive sequence family, named ‘TaiI family’, which consisted of a repeat unit of 570 bp. Fluorescence in-situ hybridization of the chromosomes of L. racemosus indicated that both families were located in subtelomeric heterochromatin and that the 350-bp family and TaiI family occupied different heterochromatin regions. In addition, even homologous chromosomes did not show the same patterns of TaiI and 350-bp families. The combination of these two families of repetitive sequences, together with Afa-family sequences and rDNAs, helps to identify the ten homologous chromosome pairs of L. racemosus. From these data, we proposed a karyotype of L. racemosus and compared it with other karyotypes already reported. This revised version was published online in August 2006 with corrections to the Cover Date.     

Micromechanical studies of mitotic chromosomes

We review micromechanical experiments on mitotic chromosomes. We focus on work where chromosomes were extracted from prometaphase amphibian cells, and then studied by micromanipulation and microfluidic biochemical techniques. These experiments reveal that chromosomes have well-behaved elastic response over a fivefold range of stretching, with an elastic modulus similar to that of a loosely tethered polymer network. Perturbation by microfluidic ‘spraying’ of various ions reveals that the mitotic chromosome can be rapidly and reversibly decondensed or overcondensed, i.e. that the native state is not maximally compacted. Finally, we discuss microspraying experiments of DNA-cutting enzymes which reveal that the element which gives mitotic chromosomes their mechanical integrity is DNA itself. These experiments indicate that chromatin-condensing proteins are not organized into a mechanically contiguous ‘scaffold’, but instead that the mitotic chromosome is best thought of as a cross-linked network of chromatin. Preliminary results from restriction-enzyme digestion experiments indicate a spacing between chromatin ‘cross-links’ of roughly 15 kb, a size similar to that inferred from classical chromatin-loop-isolation studies. We compare our results to similar experiments done by Houchmandzadeh and Dimitrov (J Cell Biol 145: 215–213 (1999)) on chromatids reconstituted using Xenopus egg extracts. Remarkably, while the stretching elastic response of the reconstituted chromosomes is similar to that observed for chromosomes from cells, the reconstituted chromosomes are far more easily bent. This result suggests that reconstituted chromatids have a large-scale structure which is quite different from chromosomes in somatic cells. More generally our results suggest a strategy for the use of micromanipulation methods for the study of chromosome structure. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distribution of L1-retroposons on the giant sex chromosomes of Microtus cabrerae (Arvicolidae, Rodentia): functional and evolutionary implications

Long interspersed nuclear elements (L1 or LINE-1) are the most abundant and active retroposons in the mammalian genome. Traditionally, the bulk of L1 sequences have been explained by the ‘selfish DNA’ hypothesis; however, recently it has been also argued that L1s could play an important role in genome and gene organizations. The non-random chromosomal distribution of these retroelements is a striking feature considered to reflect this functionality. In the present study we have cloned and analyzed three different L1 fragments from the genome of the rodent Microtus cabrerae. In addition, we have examined the chromosomal distribution of this L1 in several species of Microtus, a very interesting group owing to the presence in some species of enlarged (‘giant’) sex chromosomes. Interestingly, in all species analyzed, L1-retroposons have preferentially accumulated on both the giant- and the normal-sized sex chromosomes compared with the autosomes. Also we have demonstrated that L1-retroposons are not similarly distributed among the heterochromatic blocks of the giant sex chromosomes in M. cabrerae and M. agrestis, which suggest that L1 retroposition and amplification over the sex heterochromatin have been different and independent processes in each species. Finally, we proposed that the main factors responsible for the L1 distribution on the mammalian sex chromosomes are the heterochromatic nature of the Y chromosome and the possible role of L1 sequences during the X-inactivation process.     

Meiotic chromosomes and stages of sex chromosome evolution in fish: zebrafish, platyfish and guppy

We describe SC complements and results from comparative genomic hybridization (CGH) on mitotic and meiotic chromosomes of the zebrafish Danio rerio, the platyfish Xiphophorus maculatus and the guppy Poecilia reticulata. The three fish species represent basic steps of sex chromosome differentiation: (1) the zebrafish with an all-autosome karyotype; (2) the platyfish with genetically defined sex chromosomes but no differentiation between X and Y visible in the SC or with CGH in meiotic and mitotic chromosomes; (3) the guppy with genetically and cytogenetically differentiated sex chromosomes. The acrocentric Y chromosomes of the guppy consists of a proximal homologous and a distal differential segment. The proximal segment pairs in early pachytene with the respective X chromosome segment. The differential segment is unpaired in early pachytene but synapses later in an ‘adjustment’ or ‘equalization’ process. The segment includes a postulated sex determining region and a conspicuous variable heterochromatic region whose structure depends on the particular Y chromosome line. CGH differentiates a large block of predominantly male-specific repetitive DNA and a block of common repetitive DNA in that region. This revised version was published online in July 2006 with corrections to the Cover Date.     

Origins and inheritance of chromosome-length polymorphisms in the barley covered smut fungus, Ustilago hordei

Numerous chromomsome-length polymorphisms (CLPs) associated with chromosome IV were detected in an inbred line of race 8 of Ustilago hordei (teliospore line 1279). Polymorphisms for chromosome IV were observed in the 1600–1900-kb range in approximately 8% of the haploid sporidia originating from this teliospore collection. A monosporidial strain, 1279Ca2, exhibited a new 1620-kb chromosome band and a concurrent loss of the 1950-kb chromosome IV. A ribosomal DNA probe from Armillaria mellea specifically hybridized to both the variant 1620-kb chromosome and to the 1950-kb rDNA chromosome IV in parental strains. Following digestion of chromosome IV with HinfI, the telomere fragments of the 1620-kb chromosome were similar to those of the 1950-kb chromosome IV, indicating that the 1620-kb chromosome arose following a deletion of approximately 330 kb from chromosome IV. The Hinf1 digest of chromosome IV, when probed with the rDNA probe, revealed that much of the rDNA of chromosome IV was lost in the 1279Ca2 line. rDNA sequences were coincidentally recorded in chromosomes I and II in the sister sporidial line, 1279Ca4. When the 1279Ca2 line was mated to other members within the same tetrad and inoculated onto susceptible barley, karyotypes of tetrads and random sporidia originating from the F₁ progeny possessed variant chromosomes ranging in size from 1536 to 2110 kb. Among sporidia in 5 of the 12 ordered tetrads sampled, a 1:1 ratio of the 1950-kb parental chromosome IV to the variant chromosome was observed. Within these tetrads, the two polymorphic chromosomes were identical in size and larger than that of the original variant 1620-kb chromosome suggesting that a chromosome expansion, averaging 150 kb, had occurred. In 5 of the 12 tetrads, a 1:1:1:1 ratio representing the two original parents and two recombinant chromosomes was observed, suggesting that normal or unequal recombination had occurred during meiosis. In 2 of the 12 F₁ progeny tetrads, the 1950-kb chromosome IVs were apparently eliminated. In karyotypes of these sporidial lines, we observed rDNA sequences in chromosomes I and III that were translocated from chromosome IV. Among 78 random sporidia in the F₁ generation, duplication of the variant chromosome IV was observed in three strains. These results suggest that polymorphisms in the rDNA chromosome IV, which consist of chromosome expansion, translocation, and chromosome elimination or duplication, are common in the 1279 strain of U. hordei and its progeny, and that this variability appears to be associated with the tandem-repeat nature of the rDNA sequences. Received: 12 May / 18 November 1997     

Compositional mapping of chicken chromosomes and identification of the gene-richest regions

Compositional chromosomal mapping, namely the assessment of the GC level of chromosomal bands, led to the identification, in the human chromosomes, of the GC-richest H3⁺ bands and of the GC-poorest L1⁺ bands, which were so called on the basis of the isochore family predominantly present in the bands. The isochore organization of the avian genome is very similar to those of most mammals, the only difference being the presence of an additional, GC-richest, H4 isochore family. In contrast, the avian karyotypes are very different from those of mammals, being characterized, in most species, by few macrochromosomes and by a large number of microchromosomes. The compositional mapping of chicken mitotic and meiotic chromosomes by in-situ hybridization of isochore families showed that the chicken GC-richest isochores are localized not only on a large number of microchromosomes but also on almost all telomeric bands of macrochromosomes. On the other hand, the GC-poorest isochores are generally localized on the internal regions of macrochromosomes and are almost absent in microchromosomes. Thus, the distinct localization of the GC-richest and the GC-poorest bands observed on human chromosomes appears to be a general feature of chromosomes from warm-blooded vertebrates.     

Molecular cytogenetic characterization of the EBV-producing cell line B95-8 (Saguinus oedipus, Platyrrhini) by chromosome sorting and painting

The cell line B95-8 releases Epstein–Barr virus (EBV) with high titres of transforming activity and is widely used as a model in cancer research and virology. There are, however, controversial reports about the species of origin, cell line stability and karyotype. To address these questions, B95-8 chromosomes were analysed by chromosome sorting and painting by multicolour fluorescence in-situ hybridization. Reciprocal painting was performed between B95-8, ‘wildtype’ New World monkey and human chromosomes. Saguinus oedipus was revealed as the species of origin. A further five cell-line-specific marker chromosomes, resulting from translocations, deletions and an insertion were found. Although human chromosome 6 or 13 homologues were always involved in these rearrangements, co-hybridization of an EBV-specific DNA probe did not reveal site-specific hybridization to marker chromosomes or at translocation breakpoints. The multicolour probe set described here will be of special value for further evolutionary studies in New World monkeys. This revised version was published online in July 2006 with corrections to the Cover Date.