Multicolour spectral karyotyping of mouse chromosomes.

Murine models of human carcinogenesis are exceedingly valuable tools to understand genetic mechanisms of neoplastic growth. The identification of recurrent chromosomal rearrangements by cytogenetic techniques serves as an initial screening test for tumour specific aberrations. In murine models of human carcinogenesis, however, karyotype analysis is technically demanding because mouse chromosomes are acrocentric and of similar size. Fluorescence in situ hybridization (FISH) with mouse chromosome specific painting probes can complement conventional banding analysis. Although sensitive and specific, FISH analyses are restricted to the visualization of only a few mouse chromosomes at a time. Here we apply a novel imaging technique that we developed recently for the visualization of human chromosomes to the simultaneous discernment of all mouse chromosomes. The approach is based on spectral imaging to measure chromosome-specific spectra after FISH with differentially labelled mouse chromosome painting probes. Utilizing a combination of Fourier spectroscopy, CCD-imaging and conventional optical microscopy, spectral imaging allows simultaneous measurement of the fluorescence emission spectrum at all sample points. A spectrum-based classification algorithm has been adapted to karyotype mouse chromosomes. We have applied spectral karyotyping (SKY) to chemically induced plasmocytomas, mammary gland tumours from transgenic mice overexpressing the c-myc oncogene and thymomas from mice deficient for the ataxia telangiectasia (Atm) gene. Results from these analyses demonstrate the potential of SKY to identify complex chromosomal aberrations in mouse models of human carcinogenesis.

The promise of comparative genomics in mammals.

Dense genetic maps of human, mouse, and rat genomes that are based on coding genes and on microsatellite and single-nucleotide polymorphism markers have been complemented by precise gene homolog alignment with moderate-resolution maps of livestock, companion animals, and additional mammal species. Comparative genetic assessment expands the utility of these maps in gene discovery, in functional genomics, and in tracking the evolutionary forces that sculpted the genome organization of modern mammalian species.

Multicolor spectral karyotyping of human chromosomes.

The simultaneous and unequivocal discernment of all human chromosomes in different colors would be of significant clinical and biologic importance. Whole-genome scanning by spectral karyotyping allowed instantaneous visualization of defined emission spectra for each human chromosome after fluorescence in situ hybridization. By means of computer separation (classification) of spectra, spectrally overlapping chromosome-specific DNA probes could be resolved, and all human chromosomes were simultaneously identified.

Chromosome painting in mammals as an approach to comparative genomics.

Chromosome painting has become a routine tool in comparative cytogenetics. The utility of interspecies chromosome painting has been demonstrated in taxa characterized by highly rearranged karyotypes such as in rodents and lesser apes. Chromosome painting also provides a new level of precision in comparative genome analysis for eliminating errors of confounding convergence with homology. Recent results hold promise that molecular cytogenetics will make a significant contribution to the understanding of the major features of genome evolution.

Comparative painting of mammalian chromosomes.

Comparative chromosome painting has shown that synteny has been conserved for large segments of the genome in various placental mammals. Advances such as spectral karyotyping and multicolour 'bar coding' lend speed and precision to comparative molecular cytogenetics. Reciprocal chromosome painting and hybridizations with probes such as yeast artificial chromosomes, cosmids, and fibre fluorescence in situ hybridisation allow subchromosomal assignments of chromosome regions and can identify breakpoints of rearranged chromosomes. Advances in molecular cytogenetics can now be used to test the hypothesis that chromosome rearrangement breakpoints in human pathology and in evolution are correlated.

Comparative genomics: lessons from cats.

The genomics era, spear headed by dazzling technological developments in human and mouse gene mapping, has additionally provoked extensive comparative gene mapping projects for domestic species of several vertebrate orders. As the human genome project promises a one dimensional string of 100,000 genes and sequences, comparative mapping will extend that inference to a second dimension representing index species of the 20 living mammalian orders and to a third dimension by phylogenetic description of the genomes of mammal ancestors. We review here the remarkable extent of genome homology conservation among mammals illustrated by technology applications in the feline genome project.

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.

Hepatoma-derived integrated HBV DNA causes multi-stage transformation in vitro.

The hepatoma-derived hepatitis B virus (HBV) DNA insert HU-a has recently been shown to contain two viral transactivator genes, X and preS2 /S. We report here that HU-a induces malignant transformation after stable transfection of the fetal mouse hepatocyte line FMH202, as indicated by soft agar growth and nude mouse tumorigenicity. Transfections with HU-a subclones, containing the X gene of the preS2 /S gene alone or sequences without transactivator gene, respectively, suggested that the X gene is essential for transformation. Sequential stages of transformation and tumor progression were analysed by injection of the stably transfected FMH202 lines into nude mice, explanation of the resulting tumors and re-establishment of cell lines from the tumors. Comparison of two HU-a-transformed cell lines by HBV mRNA hybridization, Southern analysis and chromosomal in situ hybridization revealed that integrated HBV DNAs were involved in major chromosomal rearrangements in both cases. Interestingly, recombination of the HBV Dna insert during the nude mouse passage had completely abolished HBV-specific transcription in one case, indicating that expression of integrated HBV genes, while presumably involved in early transformation, is dispensable at later stages of tumor progression. The sequential transformation observed in this experimental system suggests that expression of the X gene by integrated viral DNA and subsequent hepatocyte genome mutations might both contribute to HBV-associated liver carcinogenesis.

Fluorescence in situ hybridization to chromosomes as a tool to understand human and primate genome evolution.

For the last 15 years molecular cytogenetic techniques have been extensively used to study primate evolution. Molecular probes were helpful to distinguish mammalian chromosomes and chromosome segments on the basis of their DNA content rather than solely on morphological features such as banding patterns. Various landmark rearrangements have been identified for most of the nodes in primate phylogeny while chromosome banding still provides helpful reference maps. Fluorescence in situ hybridization (FISH) techniques were used with probes of different complexity including chromosome painting probes, probes derived from chromosome sub-regions and in the size of a single gene. Since more recently, in silico techniques have been applied to trace down evolutionarily derived chromosome rearrangements by searching the human and mouse genome sequence databases. More detailed breakpoint analyses of chromosome rearrangements that occurred during higher primate evolution also gave some insights into the molecular changes in chromosome rearrangements that occurred in evolution. Hardly any "fusion genes" as known from chromosome rearrangements in cancer cells or dramatic "position effects" of genes transferred to new sites in primate genomes have been reported yet. Most breakpoint regions have been identified within gene poor areas rich in repetitive elements and/or low copy repeats (segmental duplications). The progress in various molecular and molecular-cytogenetic approaches including the recently launched chimpanzee genome project suggests that these new tools will have a significant impact on the further understanding of human genome evolution.

Amplified expression constructs for human tissue-type plasminogen activator in Chinese hamster ovary cells: instability in the absence of selective pressure.

By linking an expression cassette for human tissue-type plasminogen activator (t-PA) to an amplifiable marker gene, its introduction into Chinese hamster ovary dhfr- cells and subsequent amplification with methotrexate, we have generated cell lines that overproduce the heterologous protein and contain 300-1100 copies of the expression constructs integrated into the hamster genome. We present a detailed investigation of the fate of amplified sequences in the presence and absence of selective pressure by parallel examination of three producer cell lines with respect to relevant parameters. These include the determination of t-PA production upon continuous propagation in culture, the genomic organization of the integrated expression constructs by Southern blotting, and the localization of homogeneously staining regions by in-situ hybridization with biotinylated probes and visualization by interference reflection microscopy. We conclude that in the three cell lines examined, the decrease in production of t-PA in the absence of methotrexate selection is accompanied by decreases in the number of integrated expression constructs and the size of the amplified regions, whereas all these parameters are stable when selective pressure is maintained. The instability is probably due to the head-to-tail mode of integration of the expression constructs in the hamster genome, which increases the frequency of homologous recombination between the integrated plasmids in recombination-proficient cells in the absence of selective pressure.

Screening for specific chromosome involvement in hematological malignancies using a set of seven chromosome painting probes. An alternative approach for chromosome analysis using standard FISH instrumentation.

We report the application of multi-color fluorescence in situ hydribidization (FISH) for bone marrow metaphase cell analysis of hematological malignancies using a sub-set of the human karyotype for chromosome painting. A combination of chromosome probes labeled with three haptens enabled the construction of a "painting probe" which detects seven different chromosomes. The probe was used to screen three chronic myeloid leukemia (CML) derived cell lines and ten CML patient bone marrow samples for aberrations, additional to the Ph rearrangement, that are associated with the onset of blast crisis of CML. This approach was shown to identify karyotype changes commonly seen by conventional karyotyping, and in addition revealed chromosome changes unresolved or undetected by conventional cytogenetic analysis. The seven-color painting probe provides a useful, fast, and reliable complementary tool for chromosome analysis, especially in cases with poor chromosome morphology. This is a simple approach, since the probes can be displayed in a standard red/green/blue format accessible to standard fluorescence microscopes and image-processing software. The proposed approach using panels of locus-specific probes as well as chromosome paints will be useful in all diagnostic routine environments where analysis is directed towards screening for genetic rearrangements and/or specific patterns of chromosome involvement with diagnostic/prognostic value.

DA/DAPI fluorescent bands in the chromosomes of Pan paniscus.

The fluorochrome pattern produced by DA/DAPI double staining in Pan paniscus chromosomes is reported. The location of DA/DAPI prominent bands differs from that reported for all other hominoid species. However, the pattern in the pygmy chimpanzee is most similar to that seen in Pan troglodytes. Comparison of the DA/DAPI pattern of the other hominoid species allows the construction of a proposed hominoid ancestral karyotype and a preliminary phylogenetic reconstruction of DA/DAPI bands for the great apes and man.

Cytogenetic analysis shows extensive genomic rearrangements between red howler (Alouatta seniculus, Linnaeus) subspecies.

A comparison of the G-banded karyotypes of two red howler subspecies, Alouatta seniculus arctoidea and A. s. sara, showed that they differed by at least 14 chromosomal rearrangements. Genomic reshuffling is so great that homologs between subspecies could not be found for some chromosome, while the assignment of homology for other chromosomes remains uncertain. The two red howlers, however, share an unusual X1X2Y1Y2/ X1X1X2X2 sex-chromosome system that resulted from a Y-autosome translocation, probably in a common ancestor. The great chromosomal variability resulting from rapid chromosomal evolution in howlers indicates that cytogenetic data could make an important contribution to resolving phylogenetic and conservation problems in this group of highly conspicuous New World Monkeys. © 1995 Wiley-Liss, Inc.

Nonradioactive in situ hybridization with digoxigenin labeled DNA probes.

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.

Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC.

The aim of this study is to investigate additional genetic alterations in papillary thyroid carcinomas (PTCs) with known RET/PTC rearrangements. We applied array-based comparative genomic hybridization (array CGH) to 33 PTC (20 PTC from adults, 13 post-Chernobyl PTC from children) with known RET/PTC status. Principal component analysis and hierarchical cluster analysis identified cases with similar aberration patterns. Significant deviations between tumour-groups were obtained by statistical testing (Fisher's exact test in combination with Benjamini-Hochberg FDR-controlling procedure). FISH analysis on FFPE sections was applied to validate the array CGH data. Deletions were found more frequently in RET/PTC-positive and RET/PTC-negative tumours than amplifications. Specific aberration signatures were identified that discriminated between RET/PTC-positive and RET/PTC-negative cases (aberrations on chromosomes 1p, 3q, 4p, 7p, 9p/q, 10q, 12q, 13q and 21q). In addition, childhood and adult RET/PTC-positive cases differ significantly for a deletion on the distal part of chromosome 1p. There are additional alterations in RET/PTC-positive tumours, which may act as modifiers of RET activation. In contrast, alterations in RET/PTC-negative tumours indicate alternative routes of tumour development. The data presented serve as a starting point for further studies on gene expression and function of genes identified in this study.

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.

"Bar-coding" primate chromosomes: molecular cytogenetic screening for the ancestral hominoid karyotype.

Two recently introduced multicolor FISH approaches, cross-species color banding (also termed Rx-FISH) and multiplex FISH using painting probes derived from somatic cell hybrids retaining fragments of human chromosomes, were applied in a comparative molecular cytogenetic study of higher primates. We analyzed these "chromosome bar code" patterns to obtain an overview of chromosomal rearrangements that occurred during higher primate evolution. The objective was to reconstruct the ancestral genome organization of hominoids using the macaque as outgroup species. Approximately 160 individual and discernible molecular cytogenetic markers were assigned in these species. Resulting comparative maps allowed us to identify numerous intra-chromosomal rearrangements, to discriminate them from previous contradicting chromosome banding interpretations and to propose an ancestral karyotype for hominoids. From 25 different chromosome forms in an ancestral karyotype for all hominoids of 2N=48 we propose 21. Probes for chromosomes 2p, 4, 9 and Y were not informative in the present experiments. The orangutan karyotype was very similar to the proposed ancestral organization and conserved 19 of the 21 ancestral forms; thus most chromosomes were already present in early hominoid evolution, while African apes and human show various derived changes.