Reciprocal chromosome painting shows that genomic rearrangement between rat and mouse proceeds ten times faster than between humans and cats.

Reciprocal chromosome painting between mouse and rat using complete chromosome probe sets of both species permitted us to assign the chromosomal homology between these rodents. The comparative gene mapping data and chromosome painting have a better than 90% correspondence. The reciprocal painting results graphically show that mouse and rat have strikingly different karyotypes. At least 14 translocations have occurred in the 10-20 million years of evolution that separates these two species. The evolutionary rate of chromosome translocations between these two rodents appears to be up to 10 times greater than that found between humans and cats, or between humans and chimpanzees, where over the last 5-6 million years just one translocation has occurred. Outgroup comparison shows that the mouse genome has incorporated at least three times the amount of interchromosomal rearrangements compared to the rat genome. The utility of chromosome painting was also illustrated by the assignment of two new chromosome homologies between rat and mouse unsuspected by gene mapping: between mouse 11 and rat 20 and between mouse 17 and rat 6. We conclude that reciprocal chromosome painting is a powerful method, which can be used with confidence to chart the genome and predict the chromosome location of genes. Reciprocal painting combined with gene mapping data will allow the construction of large-scale comparative chromosome maps between placental mammals and perhaps other animals.     

Comparative chromosome painting defines the high rate of karyotype changes between pigs and bovids

Human and sheep chromosome-specific probes were used to construct comparative painting maps between the pig (Suiformes), cattle and sheep (Bovidae), and humans. Various yet unknown translocations were observed that would assist in a more complete reconstruction of homology maps of these species. The number of homologous segments that can be identified with sheep probes in the pig karyotype exceeds that described previously by chromosome painting between two non-primate mammals belonging to the same order. Sheep probes painted 62 segments on pig autosomes and delineated not only translocations, but also 9 inversions. All inversions were paracentric and indicate that these rearrangements may be characteristic for chromosomal changes in suiforms. Hybridizations of all sheep painting probes to cattle chromosomes confirmed the chromosome conservation in bovids. In addition, we observed a small translocation that was previously postulated from linkage mapping data, but was not yet described by physical mapping. The chromosome painting data are complemented with a map of available comparative gene mapping data between pig and sheep genomes. A detailed table listing the comparative gene mapping data between pig and cattle genomes is provided. The reanalysis of the pig karyotype with a new generation of human paint probes provides an update of the human/pig comparative genome map and demonstrates two new chromosome homologies. Seven conserved segments not yet identified by chromosome painting are also reported. Received: 2 October 2000 / Accepted: 15 January 2001     

应用端粒区带涂染探针检测染色体微小结构重排

为了评估染色体端粒区带涂染探针在遗传诊断的应用价值,应用显微切割获得的11q、12q和22q等3个染色体端粒区涂染探针（11q23.3→qter,12q24.1→qter,22q13.1→qter）,通过荧光原位杂交技术分析两个疑有染色体末端微小易位的习惯性流产病例。结果显示,病例1和病例2分别为t(11;12)和t(11;22)长臂末端间的微小易位,结合G显带技术确定断裂位点位于11q23.3、12q24.1、22q13.1。结果表明特异性染色体端粒区带探针可以确诊染色体末端区域的微小结构异常,可作为一种检出隐匿易位携带者并确定断裂位点的方法。     

Multidirectional chromosome painting reveals a remarkable syntenic homology between the greater galagos and the slow loris

We report on the first reciprocal chromosome painting of lorisoids and humans. The chromosome painting showed a remarkable syntenic homology between Otolemur and Nycticebus. Eight derived syntenic associations of human segments are common to both Otolemur and Nycticebus, indicative of a considerable period of common evolution between the greater galago and the slow loris. Five additional Robertsonian translocations form the slow loris karyotype, while the remaining chromosomes are syntenically equivalent, although some differ in terms of centromere position and heterochromatin additions. Strikingly, the breakpoints of the human chromosomes found fragmented in these two species are apparently identical. Only fissions of homologs to human chromosomes 1 and 15 provide significant evidence of a cytogenetic link between Lemuriformes and Lorisiformes. The association of human chromosomes 7/16 in both lorisoids strongly suggests that this chromosome was present in the ancestral primate genome.     

Chromosome painting and comparative physical mapping of the sex chromosomes in <Emphasis Type="Italic">Populus tomentosa</Emphasis> and <Emphasis Type="Italic">Populus deltoides</Emphasis>

Dioecious species accounted for 6% of all plant species, including a number of crops and economically important species, such as poplar. However, sex determination and sex chromosome evolution have been studied only in few dioecious species. In poplar, the sex-determining locus was mapped to chromosome 19. Interestingly, this locus was mapped to either a peritelomeric or a centromeric region among different poplar species. We developed an oligonucleotide (oligo)-based chromosome painting probe based on the sequence of chromosome 19 from Populus trichocarpa. We performed chromosome painting in P. tomentosa and P. deltoides. Surprisingly, the distal end on the short arm of chromosome 19, which corresponds to the location of the sex-determining locus reported in several species, was not painted in both species. Thus, the DNA sequences associated with this region have not been anchored to the current chromosome 19 pseudomolecule, which was confirmed by painting of somatic metaphase chromosome 19 of P. trichocarpa. Interestingly, the unpainted distal ends of the two chromosome 19 did not pair at the pachytene stage in 22–24% of the meiotic cells in the two species, suggest that these regions from the sex chromosomes have structurally diverged from each other, resulting in the reduced pairing frequency. These results shed light on divergence of a pair of young sex chromosomes in poplar.     

Quantitative microscopy after fluorescence in situ hybridization - a comparison between repeat-depleted and non-depleted DNA probes

Complex probes used in fluorescence in situ hybridization (FISH) usually contain repetitive DNA sequences. For chromosome painting, in situ suppression of these repetitive DNA sequences has been well established. Standard painting protocols require large amounts of an unlabeled 'blocking agent', for instance Cot-1 DNA. Recently, it has become possible to remove repetitive DNA sequences from library probes by means of magnetic purification and affinity PCR. Such a 'repeat depleted library probe' was hybridized to the q-arm of chromosome 15 of human metaphase spreads and interphase cell nuclei without any preannealing by Cot-1 DNA. Apart from this, 'standard' FISH conditions were used. After in situ hybridization, microscope images were obtained comparable to those achieved with the #15q library probe prior to depletion. The images were recorded by a true color CCD camera. By digital image analysis using 'line scan' and 'area scan' procedures, the painting efficiency expressed in terms of relative fluorescence signal intensity was quantitatively evaluated. The painting efficiency using the repeat depleted probe of chromosome 15q was compared to the painting efficiency after standard FISH. The results indicate that both types of probes are compatible to a high FISH efficiency. Using equivalent probe concentrations, no significant differences were found for FISH with standard painting probes and repeat depleted painting probes.     

Comparative chromosome painting between the domestic pig (Sus scrofa) and two species of peccary, the collared peccary (Tayassu tajacu) and the white-lipped peccary (T. pecari): a phylogenetic perspective

The Suidae and the Dicotylidae (or Tayassuidae) are related mammalian families, both belonging to the artiodactyl suborder Suiformes, which diverged more than 37 million years ago. Cross-species chromosome painting was performed between the domestic pig (Sus scrofa; 2n = 38), a representative of the Suidae, and two species of the Dicotylidae: the collared peccary (Tayassu tajacu; 2n = 30) and the white-lipped peccary (T. pecari; 2n = 26). G-banded metaphase chromosomes of the two peccaries were hybridized with whole chromosome painting probes derived from domestic pig chromosomes 1-18 and X. For both peccary species, a total of 31 autosomal segments that are conserved between pig and peccary could be identified. The painting results confirm conclusions inferred from G-band analyses that the karyotypes of the collared peccary and the white-lipped peccary are largely different. The karyotypic heterogeneity of the Dicotylidae contrasts with the relative homogeneity among the karyotypes of the Suidae. For this difference between the Dicotylidae and the Suidae, a number of explanations are being postulated: 1) the extant peccaries are phylogenetically less closely related than is usually assumed; 2) the peccary genome is less stable than the genome of the pigs; and 3) special (e.g. biogeographical or biosocial) circumstances have facilitated the fixation of chromosome rearrangements in ancestral dicotylid populations.     

Amplification of the Y chromosome in three murine tumor cell lines transformed in vivo by different human prostate cancers

Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.     

Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.     

A comparative study of karyotypes of muntjacs by chromosome painting

We have used a combination of chromosome sorting, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), chromosome painting and digital image capturing and processing techniques for comparative chromosome analysis of members of the genus Muntiacus. Chromosome-specific paints from a female Indian muntjac were hybridised to the metaphase chromosomes of the Gongshan, Black, and Chinese muntjac by both single and three colour chromosome painting. Karyotypes and idiograms for the Indian, Gongshan, Black and Chinese muntjac were constructed, based on enhanced 4, 6-diamidino-2-phenylindole (DAPI) banding patterns. The hybridisation signal for each paint was assigned to specific bands or chromosomes for all of the above muntjac species. The interspecific chromosomal homology was demonstrated by the use of both enhanced DAPI banding and comparative chromosome painting. These results provide direct molecular cytogenetic evidence for the tandem fusion theory of the chromosome evolution of muntjac species.Deceased     

Multicolor chromosome bar codes

Chromosome bar codes are multicolor banding patterns produced by fluorescence in situ hybridization (FISH) with differentially labeled and pooled sub-regional DNA probes. These molecular cytogenetic tools facilitate chromosome identification and the delineation of both inter- and intra-chromosomal rearrangements. We present an overview of the various conceptual approaches which can be largely divided into two classes: Simple bar codes designed for chromosome identification and complex bar codes for high resolution aberration screening of entire karyotypes. We address the issue of color redundancy and how to overcome this limitation by complementation of bar codes with whole chromosome painting probes.     

Comparative Chromosome Painting in Mammals: Human and the Indian Muntjac (Muntiacus muntjak vaginalis)

We have used human chromosome-specific painting probes forin situhybridization on Indian muntjac (Muntiacus muntjak vaginalis,2n= 6, 7) metaphase chromosomes to identify the homologous chromosome regions of the entire human chromosome set. Chromosome rearrangements that have been involved in the karyotype evolution of these two species belonging to different mammalian orders were reconstructed based on hybridization patterns. Although, compared to human chromosomes, the karyotype of the Indian muntjac seems to be highly rearranged, we could identify a limited number of highly conserved homologous chromosome regions for each of the human chromosome-specific probes. We identified 48 homologous autosomal chromosome segments, which is in the range of the numbers found in other artiodactyls and carnivores recently analyzed by chromosome painting. The results demonstrate that the reshuffling of the muntjac karyotype is mostly due to fusions of huge blocks of entire chromosomes. This is in accordance with previous chromosome painting analyses between various Muntjac species and contrasts the findings for some other mammals (e.g., gibbons, mice) that show exceptional chromosome reshuffling due to multiple reciprocal translocation events.     

Meiosis and chromosome painting of sex chromosome systems in Ceboidea

The identity of the chromosomes involved in the multiple sex system of Alouatta caraya (Aca) and the possible distribution of this system among other Ceboidea were investigated by chromosome painting of mitotic cells from five species and by analysis of meiosis at pachytene in two species. The identity of the autosome #7 (X2) involved in the multiple system of Aca and its breakage points were demonstrated by both meiosis and chromosome painting. These features are identical to those described by Consigliere et al. [1996] in Alouatta seniculus sara (Assa) and Alouatta seniculus arctoidea (Asar). This multiple system was absent in the other four Ceboidea species studied here. However, data from the literature strongly suggest the presence of this multiple in other members of this genus. The presence of this multiple system among several species and subspecies that show high levels of chromosome rearrangements may suggest a special selective value of this multiple. The meiotic features of the sex systems of Aca and Cebus apella paraguayanus (Cap) are strikingly different at pachytene, as the latter system is similar to the sex pair of man and other primates. The relatively large genetic distances between species presently showing this multiple system suggest that its origin is not recent. Other members of the same genus should be investigated at meiosis and by chromosome painting in order to know the extent and distribution of this complex sex-chromosome system.     

Radiosensitivity of ataxia telangiectasia and Nijmegen breakage syndrome homozygotes and heterozygotes as determined by three-color FISH chromosome painting

A three-color chromosome painting technique was used to examine the spontaneous and radiation-induced chromosomal damage in peripheral lymphocytes and lymphoblastoid cells from 11 patients with ataxia telangiectasia (AT) and from 14 individuals heterozygous for an AT allele. In addition, cells from two homozygous and six obligate heterozygous carriers of mutations in the Nijmegen breakage syndrome gene (NBS) were investigated. The data were compared to those for chromosome damage in 10 unaffected control individuals and 48 cancer patients who had not yet received therapeutic treatment. Based on the well-documented radiation sensitivity of AT and NBS patients, it was of particular interest to determine whether the FISH painting technique used in these studies allowed the reliable detection of an increased sensitivity to in vitro irradiation of cells from heterozygous carriers. Peripheral blood lymphocytes and lymphoblastoid cells from both the homozygous AT and NBS patients showed the highest cytogenetic response, whereas the cells from control individuals had a low number of chromosomal aberrations. The response of cells from heterozygous carriers was intermediate and could be clearly differentiated from those of the other groups in double-coded studies. AT and NBS heterozygosity could be distinguished from other genotypes by the total number of breakpoints per cell and also by the number of the long-lived stable aberrations in both AT and NBS. Only AT heterozygosity could be distinguished by the fraction of unstable chromosome changes. The slightly but not significantly increased radiosensitivity that was found in cancer patients was apparently due to a higher trend toward rearrangements compared to the controls. Thus the three-color painting technique presented here proved to be well suited as a supplement to conventional cytogenetic techniques for the detection of heterozygous carriers of these diseases, and may be superior method.     

Cross-species chromosome painting

Comparative genomics is an important and expanding field of research, and the genome-wide comparison of the chromosome constitution of different species makes a major contribution to this field. Cross-species chromosome painting is a powerful technique for establishing chromosome homology maps, defining the sites of chromosome fusions and fissions, investigating chromosome rearrangements during evolution and constructing ancestral karyotypes. Here the protocol for cross-species chromosome painting is presented. It includes sections on cell culture and metaphase preparation, labeling of chromosome-specific DNA, fluorescent in situ hybridization (chromosome painting) and image analysis. Cell culture and metaphase preparation can take between 1 and 2 wk depending on the cell culture. Labeling of chromosome-specific DNA is completed in 1 d. Fluorescent in situ hybridization can be completed in a maximum of 4 d.     

Single‐copy gene‐based chromosome painting in cucumber and its application for chromosome rearrangement analysis in Cucumis

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single‐copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome‐specific gene probes. This single‐copy gene‐based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis‐aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross‐species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.     

Molecular cytogenetic studies in strepsirrhine primates, dermoptera and scandentia

Since the first chromosome painting study between human and strepsirrhine primates was performed in 1996, nearly 30 species in Strepsirrhini, Dermoptera and Scandentia have been analyzed by cross-species chromosome painting. Here, the contribution of chromosome painting data to our understanding of primate genome organization, chromosome evolution and the karyotype phylogenetic relationships within strepsirrhine primates, Dermoptera and Scandentia is reviewed. Twenty-six to 43 homologous chromosome segments have been revealed in different species with human chromosome-specific paint probes. Various landmark rearrangements characteristic for each different lineage have been identified, as cytogenetic signatures that potentially unite certain lineages within strepsirrhine primates, Dermoptera and Scandentia.     

A mitotically stable marker chromosome negative for whole chromosome libraries, centromere probes and chromosome specific telomere regions: a novel class of supernumerary marker chromosome?

A two year-old child presented with mild developmental delay. On karyotype analysis, a supernumerary small marker chromosome (SMC) was found in all cells examined. This SMC was approximately the size of an isochromosome 18p, being symmetrical with a central constriction. C-banding and silver staining were negative and FISH with all chromosome-specific paints, centromere probes and telomere probes showed no hybridization to the SMC; telomere repeat sequences were however present on both arms. Comparative genomic hybridization showed no amplification of any chromosome region. Flow sorting of the SMC and reverse painting onto normal metaphase spreads showed no hybridization to any chromosome, whereas reverse painting onto the patient's own metaphases showed hybridization to the SMC only. This SMC may thus represent either a complex amplicon of different genomic regions, or a multifold amplification of a very small region, with a neocentromere comprising an active kinetochore but no alphoid DNA. Prognostic implications for the proband were difficult to assess due to the absence of reports of similar marker chromosomes in the literature.     

Mouse chromosome-specific painting probes generated from microdissected chromosomes

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosomes probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.     

Comparative genomics: tracking chromosome evolution in the family ursidae using reciprocal chromosome painting

The Ursidae family includes eight species, the karyotype of which diverges somewhat, in both chromosome number and morphology, from that of other families in the order Carnivora. The combination of consensus molecular phylogeny and high-resolution trypsin G-banded karyotype analysis has suggested that ancestral chromosomal fissions and at least two fusion events are associated with the development of the different ursid species. Here, we revisit this hypothesis by hybridizing reciprocal chromosome painting probes derived from the giant panda (Ailuropoda melanoleuca), domestic cat (Felis catus), and man (Homo sapiens) to representative bear species karyotypes. Comparative analysis of the different chromosome segment homologies allowed reconstruction of the genomic composition of a putative ancestral bear karyotype based upon the recognition of 39 chromosome segments defined by painting as the smallest conserved evolutionary unit segments (pSCEUS) among these species. The different pSCEUS combinations occurring among modern bear species support and extend the postulated sequence of chromosomal rearrangements and provide a framework to propose patterns of genome reorganization among carnivores and other mammal radiations.