Biomedical subjects

J Wienberg

Publications and source records attributed to J Wienberg.

At least 55 records · Page 3Linked to original sources

PubMed1997

A novel source of highly specific chromosome painting probes for human karyotype analysis derived from primate homologues.

We have established a series of highly specific painting probes for human acrocentric chromosomes. These chromosomes are involved in the formation of the nucleolar organizer region (NOR) and show DNA sequence homologies within their pericentric heterochromatin. To date, these chromosomes have shown considerable cross hybridization in chromosome painting experiments. Our probe set has been established from primate homologues that are not involved in the NOR in that particular species or from species in which highly repetitive sequences have undergone rapid sequence divergence. The new painting probes should be of particular value for automated microscopy, for which highly specific signals are required as they are recorded at low magnification, e.g. when scoring chromosome 21 domains in interphase nuclei.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Isolation of chromosome-specific paints from high-resolution flow karyotypes of the sheep (Ovis aries).

High-resolution bivariate flow karyotypes were obtained using fibroblast cell lines from a sheep with a normal karyotype (2n = 54), from sheep carrying Robertsonian translocation chromosomes and from sheep-hamster somatic cell hybrids. By taking advantage of the presence of chromosome polymorphisms, translocation chromosomes and sheep-hamster somatic cell hybrids, all sheep chromosomes were isolated by flow sorting. Chromosome-specific paints were generated from each sorted peak using degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The sheep chromosome present in each peak was identified by chromosome-specific microsatellite analysis of the DOP-PCR products and fluorescence in situ hybridization (FISH) onto DAPI-banded sheep metaphase chromosomes. The chromosome-specific DNA obtained in this study can be used for the production of genomic libraries and as a resource for mapping randomly cloned DNA sequences that will greatly aid the construction of genetic and physical maps in the sheep. The chromosome-specific paints will facilitate chromosome identification and contribute to the study of karyotype evolution in the sheep and related species.

Animals↗

PubMed1997

A reappraisal of the tandem fusion theory of karyotype evolution in Indian muntjac using chromosome painting.

We have tested the tandem fusion hypothesis of the origin of the Indian muntjac karyotype (2n = 6/7) by using reciprocal chromosome painting between the Indian muntjac, Chinese muntjac (n = 46) and brown brocket deer (2n = 70 + 3B) with chromosome-specific paint probes derived from flow-sorted chromosomes of these three deer species. Our results have shown that the euchromatic blocks of all chromosome arms of the brown brocket deer have been conserved apparently unchanged in number and content in the Indian muntjac. While confirming the conservation in toto of most of Chinese muntjac euchromatin in the karyotype of the Indian muntjac, we demonstrate that the synteny of chromosomes 1, 2, 3, 4 and 5 of the Chinese muntjac has been disrupted by chromosome rearrangements other than fusions. This indicates that the present karyotype of the Indian muntjac cannot be reconstructed from the hypothetical Chinese muntjac-like 2n = 46 ancestral karyotype exclusively by chromosome fusions. Furthermore, we have shown that the breakpoints of these rearrangements appear to have occurred near to the fusion points formed during the origin of the 2n = 46 karyotype of the Chinese muntjac from a 2n = 70 karyotype, which is believed to be ancestral for the family Cervidae. Moreover, we substantiate that on the Indian muntjac chromosomes, the C5 probe, which is derived from the centromeric satellite sequences of the Chinese muntjac, maps to the putative fusion points determined by comparative chromosome painting and presumably represents the remnants of ancestral centromeric sequences.

Animals↗

PubMed1997

Mapping chromosomal homology between humans and the black-handed spider monkey by fluorescence in situ hybridization.

We hybridized human chromosome-specific DNA probes to metaphases of the New World monkey Ateles geoffroyi to map the chromosomal homology between these two species. In the haploid Ateles geoffroyi karyotype the total number of signals was 51 for the 22 human autosomal probes used. Compared with Old World monkeys, the number of translocations found in the black-handed spider monkey karyotype was quite striking. The majority of these translocations are apparently Robertsonian and no reciprocal translocations were revealed. Nine autosomal human chromosome probes (11, 13, 14, 17, 18, 19, 20, 21, 22) provided only two signals each per metaphase, but six of these were translocated to subregions of different spider monkey chromosomes. The other 13 autosomal human chromosome paints (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16) provided fragmented signals. Three human probes (5, 8, 10) provided signals located on two pairs of spider monkey chromosomes. Four human paints (2, 3, 4, 12) provided hybridization signals on three pairs of chromosomes. Probes 6, 7, 15 provided six signals each on two pairs of chromosomes; probe 16 gave eight signals on two pairs of spider monkey chromosomes and probe 1 gave 12 signals on four pairs of chromosomes. The synteny between segments to human 18/8 appears to be an apomorphic ancestral condition for all New World monkeys. A synteny between regions homologous to human 16/10, 5/7 and 2/16 HSA is probably an apomorphic ancestral condition for all Cebidae. The syntenic association 3/15 and 4/1 is an apomorphic condition for the Atelinae.

Animals↗

PubMed1997

Evolution of the black muntjac (Muntiacus crinifrons) karyotype revealed by comparative chromosome painting.

The black muntjac (Muntiacus crinifrons) has an unusual karyotype of 2n = 8 in females and 2n = 9 in males. We have studied the evolution of this karyotype by hybridising chromosome-specific paints derived from flow-sorted chromosomes of the Chinese muntjac (M. reevesi, 2n = 46) to chromosomes of the black muntjac. The hybridisation pattern allowed us to infer chromosomal homologies between these two species. Tandem and centromeric fusions, reciprocal translocations, and insertions are involved in the reduction of the diploid number from 2n = 46 to 2n = 8, 9. The painting patterns further show complex chromosomal rearrangements in the male black muntjac which involve more than half the karyotype, including both sex chromosomes. Since early meiosis is reported to be normal without any visible inversion loops of the synaptonemal complex, the observed chromosomal rearrangements would lead to heterosynapsis and, therefore, leave a large fraction of the male black muntjac karyotype balanced between the two sexes.

Animals↗

PubMed1997

Conservation of human vs. feline genome organization revealed by reciprocal chromosome painting.

We employed fluorescence in situ hybridization (FISH) with probes established by flow sorting metaphase chromosomes of the domestic cat (Felis cattus, 2n = 38) to "paint" homologous segments on human chromosomes and, reciprocally, using human chromosome paints on feline metaphase preparations. The results revealed, by direct microscopic observation, widespread conservation of genome organization between the two mammalian orders and confirmed 90% of the homologous genes mapped to both species. Fourteen of 23 human chomosomes were hybridized with single cat probes, and 9 of 19 cat chromosomes were entirely labeled by a single human probe. All other chromosomes were labeled with only two or, at most, three probes of the respective species. Y-chromosome probes gave no signals. Approximately 30 syntenic segments were identified, and the number of translocations could be estimated to be on the order of one new translocation per 10 million years in the phylogenetic lines leading to human and cat. Using the principle of maximum parsimony, the primitive vs. derived human chromosome segments were identified by comparison to the feline, cattle, and pig genomes, a first step in reconstructing the evolutionary heritage of the mammalian radiations. The results suggest that reciprocal chromosome painting will help reconstruct the history of genomic changes by determining the polarity of chromosomal rearrangements and establishing the ancestral karyotype for each principle branching point in mammalian evolution.

Animals↗

PubMed1997

Reciprocal chromosome painting between human and prosimians (Eulemur macaco macaco and E. fulvus mayottensis).

We used fluorescence in situ hybridisation to delineate the homology between the human karyotype and those of two lemur species (Eulemur macaco macaco and E. fulvus mayottensis). Human and lemur chromosome-specific probes were established by bivariate fluorescence-activated flow sorting (FACS) and subsequent degenerate oligonucleotide-primed PCR (DOP-PCR). Reciprocal painting of human probes to lemur chromosomes and vice versa allowed a detailed analysis of the interchromosomal rearrangements that had occurred during the evolution of these species. The results indicate that the genomes of both species have undergone only a few translocations during more that 45 million years of lemur and human evolution. The synteny of homologs to human chromosomes 3, 9, 11, 13, 14, 17, 18, 20, 21, X, and Y was found to be conserved in the two lemur species. Taking non-primate mammals as the outgroup for primates, ancestral conditions for various primate chromosomes were identified and distinguished from derived forms. Lemur chromosome painting probes were also used for cross-species hybridization between the two lemur species. The results support an earlier assumption, made on the basis of chromosome banding, that the karyotypes of the two species have evolved exclusively by Robertsonian transformations. All probes derived from E. f. mayottensis chromosomes specific for homologs involved in rearrangements in E. m. macaco exclusively painted entire chromosome arms. The results further indicate that E. f. mayottensis most probably has a more ancestral karyotype than E. m. macaco. Probes derived from prosimians will be useful in comparing the karyotypes of other lower primates, which will improve our understanding of early primate genome evolution.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

Chromosome painting defines genomic rearrangements between red howler monkey subspecies.

We hybridized whole human chromosome-specific DNA libraries to chromosomes of two supposed subspecies of Alouatta seniculus: Alouatta seniculus sara and Alouatta seniculus arctoides. The number of hybridization signals per haploid set is 42 in A. s. sara and 43 in A. s. arctoidea; the two karyotypes differ by at least 16 chromosomal rearrangements, including numerous translocations. An unusual sex chromosome system is shared by both taxa. The sex chromosome system results from a Y translocation with a chromosome homologous to parts of human chromosome 3/15 and can be described as X1X2Y1Y2/X1X1X2X2 (male/female). Both red howlers also have microchromosomes, a highly unusual karyological trait not found in other higher primates. These microchromosomes are not hybridized by any human chromosome paint and therefore are probably composed of repetitive DNA. It is well known that New World monkeys have high karyological variability. It is probable that molecular cytogenetic analyses including chromosome painting will permit an accurate reconstruction of the phylogeny of these monkeys and help establish the ancestral karyotype for higher primates.

Alouatta↗

PubMed1996

Analysis of the organisation and localisation of the FSHD-associated tandem array in primates: implications for the origin and evolution of the 3.3 kb repeat family.

The D4Z4 locus is a polymorphic tandem repeat sequence on human chromosome 4q35. This locus is implicated in the neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD). The majority of sporadic cases of FSHD are associated with de novo DNA deletions within D4Z4. However, it is still not known how this rearrangement causes FSHD. Although the repeat contains homeobox sequences, despite exhaustive searching, no transcript from this locus has been identified. Therefore, it has been proposed that the deletion may invoke a position effect on a nearby gene. In order to try to understand the role of the D4Z4 repeat in this disease, we decided to investigate its conservation in other species. In this study, the long-range organisation and localisation of loci homologous to D4Z4 were investigated in primates using Southern blot analysis, pulsed field gel electrophoresis and fluorescence in situ hybridisation. In humans, probes to D4Z4 identify, in addition to the 4q35 locus, a closely related tandem repeat at 10qter and many related repeat loci mapping to the acrocentric chromosomes; a similar pattern was seen in all the great apes. In Old World monkeys, however, only one locus was detected in addition to that on the homologue of human chromosome 4, suggesting that the D4Z4 locus may have originated directly from the progenitor locus. The finding that tandem arrays closely related to D4Z4 have been maintained at loci homologous to human chromosome 4q35-qter in apes and Old World monkeys suggests a functionally important role for these sequences.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

Molecular characterization of breakpoints in patients with holoprosencephaly and definition of the HPE2 critical region 2p21.

Holoprosencephaly (HPE) is a common developmental defect involving the brain and face in humans. Cytogenetic deletions in patients with HPE have localized one of the HPE genes (HPE2) to the chromosomal region 2p21. Here we report the molecular genetic characterization of nine HPE patients with cytogenetic deletions or translocations involving 2p21. We have determined the parental origin of the deleted chromosomes and defined the HPE2 critical region between D2S119 and D2S88/D2S391. As a first step towards cloning the HPE2 gene which is crucial for normal brain development we have constructed a YAC contig which spans the smallest region of deletion overlap. Several of these YACs could be identified which span three different 2p21 breakpoints in HPE patients. These YACs narrow the HPE2 critical region to less than 1 Mb and are now being further analyzed to identify the gene causing holoprosencephaly on chromosome 2.

Base Sequence↗

PubMed1996

Identification of complex chromosome rearrangements in the gibbon by fluorescent in situ hybridization (FISH) of a human chromosome 2q specific microlibrary, yeast artificial chromosomes, and reciprocal chromosome painting.

Chromosome painting has revealed that the human chromosome homologs in lesser apes are often fragmented and translocated to a number of different hylobatid chromosomes. We investigated the fragmented human chromosome 2 homologs in gibbons to illustrate a new strategy in mapping regional and band-specific chromosomal homologies between species. Previous research showed that the DNA library specific to human chromosome 2 paints parts of four gibbon (lar species group) chromosomes (viz., 1, 10, 12, and 16) and yields five distinct hybridization signals (including two on gibbon chromosome 16). However, the exact segments of human chromosome 2 that were translocated to the various gibbon chromosomes could not be distinguished. To determine the origin of the human chromosome 2 signals, we hybridized a microlibrary for the long arm of human chromosome 2, as well as YACs specific for most of the major bands on this chromosome, to metaphases of the gibbon. For reciprocal chromosome painting, we hybridized flow-sorted gibbon chromosome probes to human chromosome 2. Each method added additional insights that helped clarify the shuffling of human chromosome 2 material in the highly reorganized gibbon genome. There was an excellent correspondence between these complementary techniques. YAC 958d2 identified the breakpoint between human chromosome 2 material present on gibbon chromosomes 10 and 16. The reciprocal chromosome painting permitted a more complete and regional assignment of homology between segments on various gibbon chromosomes to human chromosome 2. The results show that a combination of reciprocal chromosome painting, subregional microlibraries, and band-specific probes (such as YACs) can be used to identify homologies between species and to rapidly construct detailed comparative chromosome maps, especially when the karyotypes are highly rearranged.

Animals↗

PubMed1995

Genomic reorganization in the concolor gibbon (Hylobates concolor) revealed by chromosome painting.

We employed fluorescence in situ hybridization (FISH) of specific DNA libraries of all human chromosomes to establish homologies between the human karyotype and the karyotype of Hylobates concolor (2n = 52). Numerous intra- and interchromosomal rearrangements have led to a massive reorganization in the karyotype of H. concolor. There have been a minimum of 31 translocations. The 25 H. concolor autosomes are composed of 63-67 recognizable segments that show DNA homology to regions of the 22 human autosomes. Only 1 autosome, homologous to human chromosome 21, has not been involved in translocations. We compared the gene mapping data for H. concolor with our in situ hybridization and found that in most cases these data are confirmed by our results. H. concolor presents a case in a primate closely related to humans, in which chromosome morphology and synteny are highly disturbed in a manner similar to that encountered in rodents.

Animals↗

PubMed1995

Comparative mapping of DNA probes derived from the V kappa immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) of cosmid clones of human V kappa gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the kappa locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V kappa I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V kappa region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution.

Animals↗