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At least 19 recordsLinked to original sources
PubMed1992

Spatial distribution of sperm-derived chromatin in zygotes determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. A hybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromosome 4, for the heterochromatic region on the long arm of the human Y chromosome and for unique DNA sequences on human chromosome 19. Chromosome 4 occupied a circumscribed domain in the pronuclei, similar to findings in somatic interphases. Unlike the situation in somatic interphases, the Y heterochromatin was extended throughout the first cell cycle. Pronuclear chromatin was extended 3- to 4-fold compared to somatic interphase chromatin. The extended pronuclear chromatin conformation is likely to affect a zygote's susceptibility to environmental hazards.

Animals↗
PubMed1992

Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RB1) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 lambda phage clones spanning the whole RB1 gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q14. Normal interphase nuclei showed two RB1 signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RB1 gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RB1 gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RB1 locus. This indicates that LOH at the RB1 locus in breast cancer cells probably involves mechanisms other than physical deletion.

Breast Neoplasms↗
PubMed1992

Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization.

Fluorescence in situ hybridization (FISH) with biotin-labeled probes mapping to 11p13 has been used for the molecular analysis of deletions of the WAGR (Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation) locus. We have detected a submicroscopic 11p13 deletion in a child with inherited aniridia who subsequently presented with Wilms tumor in a horseshoe kidney, only revealed at surgery. The mother, who has aniridia, was also found to carry a deletion including both the aniridia candidate gene (AN2) and the Wilms tumor predisposition gene (WT1). This is therefore a rare case of an inherited WAGR deletion. Wilms tumor has so far only been associated with sporadic de novo aniridia cases. We have shown that a cosmid probe for a candidate aniridia gene, homologous to the mouse Pax-6 gene, is deleted in cell lines from aniridia patients with previously characterized deletions at 11p13, while another cosmid marker mapping between two aniridia-associated translocation breakpoints (and hence a second candidate marker) is present on both chromosomes. These results support the Pax-6 homologue as a strong candidate for the AN2 gene. FISH with cosmid probes has proved to be a fast and reliable technique for the molecular analysis of deletions. It can be used with limited amounts of material and has strong potential for clinical applications.

Adult↗
PubMed1992

Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization.

Fluorescent lanthanide chelates with long decay times allow the suppression of the fast decaying autofluorescence in biological specimens. This property makes lanthanide chelates attractive as labels for fluorescence microscopy. As a consequence of the suppression of the background fluorescence the sensitivity can be increased. We modified a standard epifluorescence microscope for time-resolved fluorescence imaging by adding a pulsed light source and a chopper in the narrow aperture plane. A cooled CCD-camera was used for detection and the images were digitally processed. A fluorescent europium chelate was conjugated to antisera and to streptavidin. These conjugates were used for the localization of tumor associated antigen C242 in the malignant mucosa of human colon, for the localization of type II collagen mRNA in developing human cartilaginary growth plates, and for the detection of HPV type specific gene sequences in the squamous epithelium of human cervix. The specific slowly decaying fluorescence of the europium label could be effectively separated from the fast decaying background fluorescence. It was possible to use the europium label at the cell and tissue level and the autofluorescence was effectively suppressed in in situ hybridization and immunohistochemical reactions in both frozen and formaldehyde-fixed, wax-embedded specimens.

Alkaline Phosphatase↗
PubMedDate not supplied

Using fluorescence in situ hybridization (FISH) in genome mapping.

Fluorescence in situ hybridization (FISH) provides one of the most effective and rapid approaches for assigning and ordering DNA fragments within single eukaryotic chromosome bands. These techniques have wide applications not only for the mapping of the human genome and the genomes of other organisms, but also in clinical cytogenetics, somatic cell genetics, cancer diagnosis and gene expression studies.

Chromosome Mapping↗
PubMed1992

Localization of the synovial sarcoma t(X;18)(p11.2;q11.2) breakpoint by fluorescence in situ hybridization.

A high proportion of synovial sarcomas contain a chromosome translocation t(X;18)(p11.2;q11.2). We have previously used somatic cell hybrids derived from an established cell line, SS255, to map the X chromosome breakpoint to the interval flanked by the markers DXS14 and DXS146. In this study we have examined these hybrids with thirteen additional markers located at Xp11.3-Xcen, by Southern hybridization. Based on these results we have delimited the breakpoint as follows Xpter-DXS228-(UBE1-OATL1-TIMP-DXS226 )-(DXS255-TFE3-ELK1-DXS146)-OATL2- X;18-(DXS14-DXS422-DXS423-DXS674-DXS679)-+ ++Xcen. Confirmation of the breakpoint location has been obtained by analysis of two synovial sarcoma cell lines, SS255 and HA2243, using fluorescence in situ hybridization. A 350kb YAC probe spanning the DXS423 locus hybridized only to the derivative X chromosome, showing that it maps proximal to the breakpoint. Two YAC probes of 300kb and 450kb, containing the OATL2 locus, hybridized to both derivative chromosomes, indicating that these YACs span the translocation breakpoint. Similar results were obtained with both cell lines. The identification of YACs that span the t(X;18) breakpoint now facilitates a strategy for cloning candidate genes from this precisely defined region.

Animals↗
PubMed1992

ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization.

We illustrate the use of fluorescence in situ hybridization (FISH) for analysis of ERBB2 oncogene copy number, the level of amplification (here defined as the ratio of ERBB2 copy number to copy number of chromosome 17 centromeres), and the distribution of amplified genes in breast cancer cell lines and uncultured primary breast carcinomas. The relative ERBB2 copy number determined by FISH in 10 breast cancer cell lines correlated strongly with Southern blot results (r = 0.98) when probes for an identical reference locus were used in the two methods. Metaphase analysis of cell lines showed that amplified ERBB2 copies always occurred in intrachromosomal clusters but that the number and chromosomal location of these clusters varied among the cell lines. In interphase nuclei of primary tumors showing ERBB2 amplification (10/44), ERBB2 copies were seen as one to four clusters, also suggesting intrachromosomal localization. Regardless of the average level of amplification, all these tumors contained highly amplified cell subpopulations with at least 25, and sometimes more than 100, ERBB2 copies per cell. Tumors that did not show amplification by FISH (34/44) had an average of one to five ERBB2 copies scattered randomly in the nuclei and completely lacked cells with high copy levels. FISH results on primary tumors were concordant with slot blot results on amplification and with immunohistochemical detection of overexpression. Quantitative analysis of ERBB2 amplification by FISH may improve prognostic assessments based on the pattern of amplification and detection of heavily amplified tumor cell subpopulations.

Breast Neoplasms↗
PubMed1992

Radiation-induced chromosome aberrations analysed by fluorescence in situ hybridization with a triple combination of composite whole chromosome-specific DNA probes.

Fluorescence in situ hybridization (FISH) with a combination of three composite whole chromosome-specific DNA probes for human chromosomes 1, 4 and 12 was used to analyse in vitro radiation-induced dicentrics and symmetrical translocations in peripheral lymphocytes. Translocations could be rapidly and efficiently detected by FISH. Their frequencies were 1.8-fold higher than the frequencies for dicentrics at a given dose. The dose-response curves for translocations and dicentrics were linear quadratic with a significant higher quadratic component for translocations. The application of FISH for scoring stable translocations for biological dosimetry of radiation exposures is discussed.

Adult↗
PubMed1992

Multiple colors by fluorescence in situ hybridization using ratio-labelled DNA probes create a molecular karyotype.

Fluorescence in situ hybridization (FISH) is now widely used for the localization of genomic DNA fragments, and the identification of chromosomes by painting. We now show that half of the chromosomal complement can be painted in twelve different colors by using human chromosome specific libraries carrying three distinct labels mixed in multiple ratios. The photographs are in 'real' color rather than 'colorized'. The painting technique described here can be used for the identification of small or complex chromosomal rearrangements and marker chromosomes in humans or in any other species for which well defined chromosome specific libraries exist in a laboratory equipped with a conventional fluorescence microscope. The versatility of this novel cytogenetic technology may well constitute an advancement comparable to the introduction of chromosome banding and high resolution analysis of chromosomes in prometaphase.

Chromosome Mapping↗
PubMed1992

A high-resolution cytogenetic map of human chromosome 3: localization of 291 new cosmid markers by direct R-banding fluorescence in situ hybridization.

We localized 291 new cosmid markers (including 65 RFLPs) on human chromosome 3 by direct R-banding fluorescence in situ hybridization. This system, which is based on fluorescence in situ hybridization combined with replicated prometaphase R-bands, allows the direct visualization of signals on R-banded prometaphases stained with propidium iodide and provides a more rapid and efficient method for genome mapping of cosmid clones. The signals of 291 markers examined here were localized preferentially to R-positive bands throughout chromosome 3. The detailed map positions of 366 clones and the characterization of 142 RFLPs, including the preliminary data reported by Yamakawa et al. (1991, Genomics 9: 536-543; and 11: 565-572), are summarized. This high-resolution cytogenetic map (average distance of 0.58 Mb), in conjunction with a genetic linkage map, can facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Furthermore, these mapping data will provide many useful landmarks for the construction of contig maps of chromosome 3.

Chromosome Banding↗
PubMed1992

Localization of PMP-22 gene (candidate gene for the Charcot-Marie-Tooth disease 1A) to band 17p11.2 by direct R-banding fluorescence in situ hybridization.

We mapped PMP-22 gene, candidate gene for the Charcot-Marie-Tooth disease (CMT) 1A, by direct R-banding fluorescence in situ hybridization. The signals of PMP-22 probe were localized to chromosome band 17p11.2. The present result was within the map position of the CMT 1A gene by genetic linkage analysis, and strongly indicated that PMP-22 gene is a candidate gene for the CMT 1A.

Charcot-Marie-Tooth Disease↗
PubMed1992

Oligonucleotide probes for the analysis of specific repetitive DNA sequences by fluorescence in situ hybridization.

Five non-isotopically labeled oligonucleotides have been designed and synthesized to facilitate the analysis of specific human and murine repetitive DNA sequences by fluorescence in situ hybridization (FISH). Three of the oligonucleotides contain alphoid DNA sequences; one hybridizes to the centromeres of all human and mouse chromosomes except the Y, while the other two are specific for human chromosomes 2 and 12, respectively. The fourth oligomer, containing sequences from the spacer region of a human 5S rDNA repeat, was used to confirm the map location of a approximately 100 copy 5S rDNA tandem repeat locus. The fifth oligomer, specific to the Alu family of repeats, generates a sharp R-banding pattern on human metaphase chromosomes, suitable for FISH karyotyping. These probes permit highly specific chromosome enumeration and aneuploidy detection with hybridization times as short as 30 minutes.

Animals↗
PubMedDate not supplied

Identification of sex chromosomal abnormalities by fluorescence in situ hybridization.

Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by fluorescence in situ hybridization (FISH). Case 1 was a 20 year-old female patient who had chromosomal analysis six years ago because of primary amenorrhea and short stature. Three cell lines were identified: 45, X (20%); 46, X plus an unidentifiable fragment (50%) and 46, X plus an isodicentric X chromosome of short arm fusion (30%). Case 2 was a 6 year-old girl with the problems of clitoromegaly and growth retardation. By use of in situ hybridization with biotinylated X and Y centromere-specific alpha repetitive sequence probes, we were able to identify the marker chromosomes to be X and Y-chromosome originated, respectively. The use of FISH can facilitate clinical cytogenetic diagnosis, and allows fast screening of a large number of metaphases. This technique is applicable to the screening of other types of chromosomal mosaicism as well.

Adolescent↗
PubMed1992

Deletion of chromosome 17p loci in breast cancer cells detected by fluorescence in situ hybridization.

Allelic loss of tumor suppressor genes on chromosome 17p has been implicated in the progression of breast cancer. This is in principle detectable by fluorescence in situ hybridization if the loss occurs by deletion. In order to determine if detectable deletions occur in primary breast cancer, we used dual-color hybridization with chromosome 17 pericentromeric and region-specific DNA probes to study 19 primary breast cancers. The copy numbers of 17 centromere and 17p13.1 sequences were compared with the loss of heterozygosity (LOH) for probe YNZ22 at 17p13.3 detected by restriction fragment length polymorphism. Nine of 11 cases showing LOH also showed the major population of nuclei with a deletion. The remaining two tumors with LOH were trisomic for both the centromere and 17p13.1 cosmid. In contrast, seven of eight tumors without LOH had no deletions by fluorescence in situ hybridization. These data suggest that the dominant mechanism of allelic loss at 17p in breast cancer is a physical deletion and that analysis of deletions by fluorescence in situ hybridization is a rapid and sensitive approach to studying chromosomal aberrations.

Alleles↗