Homologous and heterologous FISH painting with PARM-PCR chromosome-specific probes in mammals

Numerous loci can be amplified by PARM-PCR on 300 sorted chromosomes in low-stringency conditions (annealing at 30°C during the two first cycles) to produce a probe that can be used in FISH painting experiments. We demonstrate that, depending on the primer chosen for the amplification, patterns of different quality can be obtained. In order to design a primer that allows amplification of coding sequences, we have shown that motifs of at least seven glutamic acid repeats (GAG or GAA codons) are present in human proteins more frequently than expected. Moreover, these repeats do not correspond to triplet expansion and can be conserved between species. Using probes prepared from sorted chromosomes with (GAG)₇ primer, we were able to achieve homologous FISH painting on human, porcine, ovine, and bovine species, and bidirectional heterologous FISH painting between human and porcine species. As an example, using probes for human Chromosome (Chr) 19 and porcine Chrs 1 and 6, we clearly defined the regional homologies existing between those chromosomes. Received: 11 July 1995 / Accepted: 9 October 1995     

Molecular cytogenetic analysis and centromeric satellite organization of a novel 8;11 translocation in sheep: a possible intermediate in biarmed chromosome evolution

During analysis of genome organization in sheep (Ovis aries, 2n = 54, XY/XX), we found a novel chromosomal translocation in an animal expected to be normal, adding to the six centric fusions previously reported. The translocation was identified as t(8;11) by G-banding and was shown to be centric, involving whole chromosome arms by chromosome painting with probes for Chromosomes (Chrs) 8 and 11. Satellite I and a newly isolated satellite II clone was used to characterize the centromeric regions of both the novel and the three pairs of evolutionarily derived biarmed chromosomes. The novel t(8;11) showed satellite I proximal on both arms with satellite II covering the centromere, while the evolutionarily derived fusion leading to Chrs 2 and 3 showed the opposite configuration, not obviously derived by a simple fusion. Chr 1 has lost the satellite I hybridization patterns. The novel t(8;11) provides strong evidence for an intermediate step in evolution of the biarmed chromosomes in sheep.     

Flexible chromosome painting based on multiplex PCR of oligonucleotides and its application for comparative chromosome analyses in Cucumis

Chromosome painting is a powerful technique for chromosome and genome studies. We developed a flexible chromosome painting technique based on multiplex PCR of a synthetic oligonucleotide (oligo) library in cucumber (Cucumis sativus L., 2n = 14). Each oligo in the library was associated with a universal as well as nested specific primers for amplification, which allow the generation of different probes from the same oligo library. We were also able to generate double‐stranded labelled oligos, which produced much stronger signals than single‐stranded labelled oligos, by amplification using fluorophore‐conjugated primer pairs. Oligos covering cucumber chromosome 1 (Chr1) and chromosome 4 (Chr4) consisting of eight segments were synthesized in one library. Different oligo probes generated from the library painted the corresponding chromosomes/segments unambiguously, especially on pachytene chromosomes. This technique was then applied to study the homoeologous relationships among cucumber, C. hystrix and C. melo chromosomes based on cross‐species chromosome painting using Chr4 probes. We demonstrated that the probe was feasible to detect interspecies chromosome homoeologous relationships and chromosomal rearrangement events. Based on its advantages and great convenience, we anticipate that this flexible oligo‐painting technique has great potential for the studies of the structure, organization, and evolution of chromosomes in any species with a sequenced genome.     

PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosomes

Whole-chromosome painting probes (WCPs) and chromosome-arm painting probes (CAPs) are an integral part of the cytogenetic analysis of chromosome abnormalities. While these are routinely made by chromosome microdissection, multiple copies of the dissected region have been necessary to achieve a library sufficiently complex to provide adequate painting. Performing multiple dissections of chromosomes or chromosome regions is time consuming and occasionally impossible, such as when working with species whose banded karyotype is not well defined. We have developed a method whereby chromosome paints can be reliably generated by dissecting single chromosomes. The technique consists of performing degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) in situ on the chromosomes, prior to dissection. Enough amplification occurs to enable a single dissected chromosome to be used to create a painting probe sufficiently complex for use in fluorescence in situ hybridization (FISH). The amplification products remain localized on the chromosomes; this allows region-specific chromosome paints to be made. We detail this novel technique and show whole-chromosome, arm-specific, and contiguous region-specific probes for human and rat, each created from single dissected fragments of chromatin. Received: 14 January 1999 / Accepted: 28 January 1999     

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.     

Ts65Dn -- localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome

Fluorescent in situ hybridization (FISH) -- using mouse chromosome paints, probes for the mouse major centromeric satellite DNA, and probes for genes on chromosomes (Chr) 16 and 17 -- was employed to locate the breakpoint in a translocation used to produce a mouse model for Down syndrome. The Ts65Dn trisomy is derived from the reciprocal translocation T(16;17)65Dn. The Ts65Dn mouse carries a marker chromosome containing the distal segment of Chr 16, a region that shows linkage conservation with human Chr 21, and the proximal end of Chr 17. This chromosome confers trisomy for most of the genes in the Chr 16 segment and Ts65Dn mice show many of the phenotypic features characteristic of Down syndrome. We used FISH on metaphase chromosomes from translocation T65Dn/+ heterozygotes and Ts65Dn mice to show that the Chr 17 breakpoint is distal to the heterochromatin of Chr 17, that the Ts65Dn marker chromosome contains a small portion of Chr 17 euchromatin, that the Chr 16 breakpoint lies between the Ncam2 and Gabpa/App genes, and that the Ts65Dn chromosome contains >80% of the human Chr 21 homologs. The significance of this finding is discussed in terms of the utility of this mouse model.     

Molecular characterization of the translocation breakpoints in the Down syndrome mouse model Ts65Dn

Ts65Dn is a mouse model of Down syndrome: a syndrome that results from chromosome (Chr) 21 trisomy and is associated with congenital defects, cognitive impairment, and ultimately Alzheimer's disease. Ts65Dn mice have segmental trisomy for distal mouse Chr 16, a region sharing conserved synteny with human Chr 21. As a result, this strain harbors three copies of over half of the human Chr 21 orthologs. The trisomic segment of Chr 16 is present as a translocation chromosome (Mmu17(16)), with breakpoints that have not been defined previously. To molecularly characterize the Chrs 16 and 17 breakpoints on the translocation chromosome in Ts65Dn mice, we used a selective enrichment and high-throughput paired-end sequencing approach. Analysis of paired-end reads flanking the Chr 16, Chr 17 junction on Mmu17(16) and de novo assembly of the reads directly spanning the junction provided the precise locations of the Chrs 16 and 17 breakpoints at 84,351,351 and 9,426,822?bp, respectively. These data provide the basis for low-cost, highly efficient genotyping of Ts65Dn mice. More importantly, these data provide, for the first time, complete characterization of gene dosage in Ts65Dn mice.     

Assignment of the rat genes coding for DOPA decarboxylase (DDC) and glutamic acid decarboxylases (GAD1 and GAD2)

By use of rat cDNA probes and a panel of cell hybrids segregating rat chromosomes, the genes encoding three pyridoxal 5-phosphate (PLP)-dependent decarboxylases—namely, DOPA-decarboxylase (Ddc), glutamic acid decarboxylase 1 and 2 (Gad1 and Gad2)—were assigned to rat Chromosomes (Chrs) 14, 3, and 17, respectively. If one takes into account chromosome localizations in the human and the mouse, the present results (i) show that a synteny group is retained on rat Chr 14, human Chr 7, and mouse Chr 11 (Ddc); (ii) strengthen the homology relation known between rat Chr 3 and human and mouse Chrs 2 (Gad1); (iii) suggest that rat Chr 17 has no extensive homology to any human chromosome; and (iv) suggest the order (Prl, Fdp)-Tpl2-Gad2 on the rat Chr 17.     

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.     

New concepts to improve resolution and sensitivity of molecular cytogenetic diagnostics by multicolor fluorescence in situ hybridization

BACKGROUND: Routine application of multicolor fluorescence in situ hybridization (M-FISH) technology for molecular cytogenetic diagnostics has been hampered by several technical limitations. First, when using chromosome-specific painting probes, there is a limit in cytogenetic resolution of approximately 2-3 Mb, which can mask hidden structural abnormalities that have a significant clinical effect. Second, using whole chromosome painting probes, intrachromosomal rearrangements cannot be detected and the exact localization of breakpoints is often not possible. METHODS: We suggest the use of multiplex-labeled region or locus- specific probes in combination with an optimal probe design to improve the sensitivity and resolution of the M-FISH technology. To allow the application of this assay in routine diagnostics, we developed a multipurpose image analysis system. RESULTS: goldFISH was applied to the study of cryptic translocations in mental retardation patients and to the study of high-resolution breakpoint mapping in non-small cell lung cancer patients. For an individual with mental retardation, who had an apparently normal karyotype by G-banding, we detected an unbalanced translocation involving chromosomes 2 and 7. CONCLUSIONS: In combination with optimally designed probe kits, goldFISH overcomes most of the present limitations of the M-FISH technology and results in virtually 100% reliability for detecting interchromosomal and intrachromosomal rearrangements.     

Integration of cytogenetic with recombinational and physical maps of mouse chromosome 16.

To link the cytogenetic map for mouse chromosome 16 (Chr 16) to the more detailed recombinational and physical maps, multiple probes were mapped by fluorescence in situ hybridization (FISH). Sixteen large insert clones (YACs, BACs, and PACs) containing markers that have been assigned to the Chr 16 recombinational map were localized to a cytogenetic band or subband by high-resolution FISH. This linkage of the cytogenetic and recombinational maps provides a useful tool for assigning new probe locations and for defining breakpoints in mice with chromosomal rearrangements. A direct application of these probes is demonstrated by identifying mice trisomic for distal Chr 16 using FISH analysis of interphase nuclei.     

Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting

A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q-;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases.     

Zoo-FISH with region-specific paints for mink chromosome 5q: delineation of inter- and intrachromosomal rearrangements in human, pig, and fox

Comparison of evolutionarily conserved mammalian chromosomes homologous to human chromosome 17, performed with microdissected painting probes, revealed rearrangements inside these chromosomes in mink and pig and a disruption of this conserved region in the fox. Detection of a homologous region on an Iberian shrew chromosome showed the efficiency of microdissected painting probes for delineation of homologous chromosome regions in species belonging to orders that diverged at least 100 million years ago.     

The genes of the lysosomal cysteine proteinases cathepsin B, H, L, and S map to different mouse chromosomes

The mouse genes for the lysosomal cysteine proteinases cathepsin B, H, L, and S were mapped to Chromosomes (Chrs) 14, 9, 13, and 3, respectively. Two of the DNA probes used in this study detected an additional, independently segregating locus. The cathepsin B-specific probe hybridized to a locus on Chr 2, and the cathepsin H probe to a locus on the X Chr. These loci either correspond to pseudogenes or to cathepsin B- and cathepsin H-related genes. The four cysteine proteinases mapped in this study lie within known regions of conserved synteny between mouse and human chromosomes, when compared with the corresponding positions of their human homologs. Assuming that the genes of the cysteine proteinase gene family arose from a common ancestral gene, our results suggest that these four cysteine proteinases had been dispersed over different chromosomes before separation of mouse and human in evolution. Received: 22 August 1996 / Accepted: 20 November 1996     

利用微分离黑麦1R染色体的体外扩增产物进行染色体着染研究

首先对显微分离出的黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体进行了两轮Ｓａｕ３Ａ连接接头介导的ＰＣＲ扩增（ＬＡ＿ＰＣＲ）。经Ｓｏｕｔｈｅｒｎ杂交证实这些染色体扩增片段来源于基因组ＤＮＡ之后,再利用１Ｒ染色体的第二轮扩增产物、黑麦基因组ＤＮＡ、ｒＤＮＡ基因为探针,与其根尖细胞中期分裂相进行染色体原位杂交,发现微分离的１Ｒ染色体体外扩增产物中包含大量的非该染色体特异性重复序列,而其信息量却较黑麦总基因组少;当以适量的黑麦基因组ＤＮＡ进行封阻时,微分离染色体的体外扩增产物成功地被重新定位在中期分裂相的一对１Ｒ染色体上,说明微分离１Ｒ染色体的ＰＣＲ扩增产物中的确包含了该染色体特异性的片段。此外,以从１Ｒ染色体微克隆文库中筛选出的一单、低拷贝序列和一高度重复序列分别为探针,染色体原位杂交检测发现,这一高度重复序列可能为端粒相关序列;而单、低拷贝序列却未检测到杂交信号。这些结果从不同侧面反映出染色体着染技术是证实微分离、微切割染色体的真实来源及筛选染色体特异性探针的有利工具。建立了可供参考的植物染色体着染实验体系,为染色体微克隆技术在植物中的进一步应用提供了便利。     

Spectral karyotyping analysis of human and mouse chromosomes

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.     

Paint-assisted microdissection-FISH: Rapid and simple mapping of translocation breakpoints in the embryonal rhabdomyosarcoma cell line RD.

BACKGROUND: Spectral karyotyping and multiple fluorophore fluorescence in situ hybridisation (M-FISH) facilitate identification of inter-chromosomal rearrangements, but are of low cytogenetic resolution in mapping translocation breakpoints. Reverse chromosome painting yields increased cytogenetic information but isolation of aberrant chromosomes is technically difficult. We have developed the technique of paint-assisted microdissection FISH (PAM-FISH), which enables microdissection of aberrant chromosomes to be carried out easily and rapidly using relatively simple apparatus. METHODS: A selected chromosome paint is hybridised to abnormal metaphases to label a chromosome of interest, which is then microdissected, amplified, labelled by polymerase chain reaction (PCR), and reverse painted onto extended normal metaphases. RESULTS: PAM-FISH was used to reassess structural chromosomal abnormalities identified by molecular cytogenetics in the rhabdomyosarcoma cell line RD. PAM-FISH improved the analysis of virtually all structural abnormalities, identifying six novel translocations and indicating that seven previously described rearrangements were in fact not present in RD. Accuracy of the breakpoint mapping obtained was confirmed by bacterial artificial chromosome-FISH. CONCLUSIONS: PAM-FISH is ideally suited to analysis of tumour metaphases as it is not affected by poor chromosome morphology. Reagents generated by PAM-FISH are also suitable for other investigations, such as mapping using sequence tagged-site PCR or genomic microarrays. PAM-FISH is technically straightforward and could readily be adopted in a routine cytogenetics laboratory for accurate high-throughput analysis of chromosome breakpoints.     

Chromosome rearrangements in canine fibrosarcomas

We have previously reported the use of six- and seven-color paint sets in the analysis of canine soft tissue sarcomas. Here we combine this technique with flow sorting of translocation chromosomes, reverse painting, and polymerase chain reaction (PCR) analysis of the gene content of the reverse paint in order to provide a more detailed analysis of cytogenetic abnormalities in canine tumors. We examine two fibrosarcomas, both from female Labrador retrievers, and show abnormalities in chromosomes 11 and 30 in both cases. Evidence of involvement of TGFBR1 is presented for one tumor.     

De novo balanced complex chromosome rearrangement (CCR) involving chromosome 8, 11 and 16 in a boy with mild developmental delay and psychotic disorder

Congenital Complex Chromosome rearrangements (CCRs) compatible with life are rare in humans. We report a de novo CCR involving chromosomes 8, 11 and 16 with 4 breakpoints in a patient with mild dysmorphic features, acquisition delay and psychotic disorder. Conventional cytogenetic analysis revealed an apparently balanced 8;16 translocation. Further FISH analysis with WCP 8 and WCP 16 probes revealed the presence of a third chromosome involved in the translocation. The multicolour karyotype confirmed the complexity of the rearrangement and showed that the derivative chromosome 8 was composed of 3 distinct segments derived from chromosomes 8, 16 and 11. The breakpoints of this complex rearrangement were located at 8q21, 11q14, 11q23 and 16q12. Comparative genomic hybridization (CGH) and array-CGH were performed to investigate the possibility of any genomic imbalance as a result of the complex rearrangement. No imbalance was detected by these two techniques. Our study showed: i) the necessity to confirm reciprocal translocations with FISH using painting probes, particularly when the karyotype resolution is weak; ii) the usefulness of multicolour karyotype for the characterization of structural chromosomal rearrangements, particularly when they are complex; iii) the usefulness of CGH and array-CGH in cases of abnormal phenotype and apparently balanced rearrangement in order to explore the breakpoints and to detect additional imbalances.     

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。