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1.
The presence of a de novo supernumerary marker chromosome (SMC) poses problems in genetic counseling. The consequences of the additional chromosomal material may range from harmless to detrimental. As the composition of a SMC cannot be deciphered by traditional banding analysis, sophisticated methods are needed for their rapid and detailed analyses. A new strategy is presented, which allows the elucidation of the composition of SMCs in one or two hybridizations. One hybridization, termed AcroM-FISH, involves a newly generated probe mix, which consists of painting probes for all acrocentric chromosomes, centromere probes for chromosomes 13/21, 14/22, 15, and a probe specific for rDNA, each labeled with a specific combination of fluorochromes. This probe mix is sufficient to characterize approximately 80% of all SMCs. For the other 20% of SMCs, chromosomes can be analyzed in a second hybridization by multicolor karyotyping, for example, multiplex FISH (M-FISH), to check for the presence of euchromatin of other chromosomes. The potential of AcroM-FISH was tested in various applications. 相似文献
2.
F. Bigoni R. Stanyon U. Koehler A. M. Morescalchi J. Wienberg 《American journal of primatology》1997,42(4):289-298
We used in situ hybridization of chromosome specific DNA probes (“chromosome painting”) of all human chromosomes to establish homologies between the human and the white and black colobus (Colobus guereza 2n = 44). The 24 human paints gave 31 signals on the autosomes (haploid male chromosome set). Robertsonian translocations between chromosomes homologus to human 14 and 15, 21 and 22, form colobine chromosomes 6 and 16, respectively. Reciprocal translocations were found between human chromosomes 1 and 10, 1 and 17, as well as 3 and 19. The alternating hybridization signals between human 3 and 19 on Colobus chromosome 12 show that in this case a reciprocal translocation was followed by a pericentric inversion. The hybridization data show that in spite of the same diploid number and similar Fundamental Numbers, the black and white colobine monkey differs from Presbytis cristata, an Asian colobine, by 6 reciprocal translocations. Comparisons with the hybridization patterns in other primates show that some Asian colobines have a more derived karyotype with respect to African colobines, macaques, great apes, and humans. Chromosome painting also clearly shows that similarities in diploid number and chromosome morphology both between colobines and gibbons are due to convergence. Am. J. Primatol. 42:289–298, 1997. © 1997 Wiley-Liss, Inc. 相似文献
3.
In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure 总被引:1,自引:0,他引:1
A. C. Van Prooijen-Knegt J. F. M. Van Hoek J. G. J. Bauman P. Van Duijn I. G. Wool M. Van der Ploeg 《Experimental cell research》1982,141(2):397-407
In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results. 相似文献
4.
Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8. 相似文献
5.
R F Suijkerbuijk D Matthopoulos L Kearney S Monard S Dhut F E Cotter J Herbergs A G van Kessel B D Young 《Genomics》1992,13(2):355-362
A novel approach to the identification of human chromosomes has been developed. Chromosomal in situ hybridization (or "chromosome painting") has been performed using Alu element-mediated PCR products from small quantities (250-500) of flow-sorted normal and abnormal chromosomes. Chromosome paints for various normal chromosomes, including 5, 6, 7, 14, 18, 19, 21, and 22, were generated and shown to be effective in the identification of the appropriate chromosomes. In addition, certain abnormal chromosomes, including a mental retardation-associated deletion chromosome 11 (q22-q23), the products of the constitutional translocation t(11;22), and the CML-associated t(9;22), were used to generate region-specific paints. In each case, the appropriate regions of the chromosomes were highlighted and this strategy is, therefore, well suited to the identification of previously unidentified marker chromosomes. A further direct consequence of this work is that chromosome paints specific for the common aberrant chromosomes, such as the Philadelphia chromosome, can be generated and made widely available. These may find particular use in the analysis of complex or masked chromosomal translocations. 相似文献
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7.
Combined GTG-banding and nonradioactive in situ hybridization improves characterization of complex karyotypes 总被引:1,自引:0,他引:1
V T Smit J W Wessels P Mollevanger P I Schrier A K Raap G C Beverstock C J Cornelisse 《Cytogenetics and cell genetics》1990,54(1-2):20-23
Nonradioactive in situ hybridization (ISH) using biotinylated centromere probes for chromosomes 1, 6, 7, 10, 16, 17, 18, and the X, respectively, was combined with GTG-banding to study cytogenetic changes in two different ovarian cancer cell lines. ISH was performed after GTG-banding on the same metaphase. The use of a low trypsin concentration (0.01%) in the banding procedure was essential for subsequent ISH. This combined approach allows the detection of subtle chromosomal rearrangements and appears to aid the identification of marker chromosomes. 相似文献
8.
DNA, chromosomes, and in situ hybridization. 总被引:6,自引:0,他引:6
Trude Schwarzacher 《Génome》2003,46(6):953-962
In situ hybridization is a powerful and unique technique that correlates molecular information of a DNA sequence with its physical location along chromosomes and genomes. It thus provides valuable information about physical map position of sequences and often is the only means to determine abundance and distribution of repetitive sequences making up the majority of most genomes. Repeated DNA sequences, composed of units of a few to a thousand base pairs in size, occur in blocks (tandem or satellite repeats) or are dispersed (including transposable elements) throughout the genome. They are often the most variable components of a genome, often being species and, occasionally, chromosome specific. Their variability arises through amplification, diversification and dispersion, as well as homogenization and loss; there is a remarkable correlation of molecular sequence features with chromosomal organization including the length of repeat units, their higher order structures, chromosomal locations, and dispersion mechanisms. Our understanding of the structure, function, organization, and evolution of genomes and their evolving repetitive components enabled many new cytogenetic applications to both medicine and agriculture, particularly in diagnosis and plant breeding. 相似文献
9.
Chromosome detection by in situ hybridization in cancer cell populations which were flow cytometrically sorted after immunolabeling. 总被引:1,自引:0,他引:1
We examined the feasibility of performing non-radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly-l-lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling. 相似文献
10.
Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes 总被引:1,自引:1,他引:0
Summary A technique of in situ hybridization on metaphase chromosomes with biotinylated DNA probes is described. This technique was used to localize unique DNA sequences on chromosomes and allowed a localization of two probes 1.8 and 1.3 kb long. The hybridization signal appears like two, twin, spots on the two sister chromatids, allowing a clear distinction from the background. Moreover a chromosomal localization is possible by counting a relatively small number of mitoses compared with the technique using 3H-labeled DNA probes. 相似文献
11.
Using a cytoplasmic actin cDNA probe we have localized a number of actin sequences in the human genome using a novel in situ hybridization technique. Metaphase chromosomes treated to produce R-bands were directly annealed with 125I-labeled actin probe. Under these conditions many regions of the genome were apparently denatured enough to be capable of hybridizing with the probe. Most of the actin sites detected in prior experiments using chromosome preparations, which had been completely denatured, were recognized in this experiment. The major advantage of this method over standard in situ hybridization techniques is the marked increase in the resolution of subregional localization. 相似文献
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Chromosomes of the pea (Pisum sativum L.) were submitted to fluorescent in situ hybridization (FISH) with probes specific for the oligonucleotides (AG)12, (AC)12, (GAA)10, and (GATA)7 and for the genes encoding 25S rRNA, 5S rRNA and the storage proteins legumin A, K and vicilin. A fourth 5S rRNA gene locus,
apparently specific for an accession of the cultivar Grüne Victoria, was newly detected. This allowed all seven chromosome
pairs to be distinguished by FISH signals of rRNA genes. The same was possible using a combination of oligonucleotide probes
or of oligonucleotides and rRNA gene-specific probes in multicolour FISH. Rehybridization with the 5S rRNA gene-specific probe
allowed us to assign vicilin genes to the short arm of chromosome 5, the single legumin A locus to the long arm of chromosome
3 and the legumin B-type genes (exemplified by legumin K) to one locus on the short arm of chromosome 6. Correlation of these
data with an updated version of the pea genetic map allowed the assignment of most linkage groups to defined chromosomes.
It only remains to be established which of linkage groups IV and VII corresponds to the satellited chromosomes 4 or 7, respectively.
Received: 13 February 1998; in revised form: 3 April 1998 / Accepted: 7 April 1998 相似文献
14.
Analysis of genes and chromosomes by nonisotopic in situ hybridization 总被引:12,自引:0,他引:12
P Lichter A L Boyle T Cremer D C Ward 《Genetic analysis, techniques and applications》1991,8(1):24-35
Nonisotopic in situ hybridization is a powerful tool to analyze the organization of complex genomes. Current approaches utilizing this technique for the analysis of linear and spatial genome organizations are presented. Clinical applications of these approaches, which open new avenues for diagnosis of disease-related chromosomal changes, are also discussed. 相似文献
15.
The identification of flow-sorted chromosomes is a very important tool for checking the purity of the fractions obtained. An easy and reproducible method for obtaining G-banded chromosomes with good resolution of bands is described. Also, we are able to show that the percentage of chromosomes which can be clearly distinguished by this procedure depends to a large extent on the duration of mitotic arrest. In particular when sorting chromosomes from human-rodent hybrid cell lines, the possibility of using in situ hybridization in addition to conventional staining techniques to characterize the chromosomes can help overcome the problem of highly condensed chromosomes and chromosomal fragments of unknown origin, which cannot be identified otherwise. Thus, we have developed an in situ hybridization technique, based on biotin-labelled human genomic DNA, which allows a clear distinction between human and rodent chromosomal material to be made. 相似文献
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17.
Summary A method is described for producing fluorescent bands on human chromosomes by staining with quinacrine after hybridisation in situ. The advantages of the method include the elimination of artefacts arising from staining before hybridisation, the fact that there is no reduction in sample number between staining and autoradiography, the ease with which autoradiographic grains can be identified and counted, and the reduction in exposure time.Offprint requests to: S.S. Lawrie 相似文献
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Meiosis,recombination and chromosomes: a review of gene isolation and fluorescent in situ hybridization data in plants 总被引:8,自引:0,他引:8
Schwarzacher T 《Journal of experimental botany》2003,54(380):11-23
Evidence is now increasing that many functions and processes of meiotic genes are similar in yeast and higher eukaryotes. However, there are significant differences and, most notably, yeast has considerably higher recombination frequencies than higher eukaryotes, different cross-over interference and possibly more than one pathway for recombination, one late and one early. Other significant events are the timing of double-strand breaks (induced by Spo11) that could be either cause or consequence of homologous chromosome synapsis and SC formation depending on the organisms, yeast plants and mammals versus Drosophila melanogaster and Caenorhabditis elegans. Many plant homologues and heterologues to meiotic genes of yeast and other organisms have now been isolated, in particular in Arabidopsis thaliana, showing that overall recombination genes are very conserved while synaptonemal complex and cohesion proteins are not. In addition to the importance of unravelling the meiotic processes by gene discovery, this review discusses the significance of chromatin packaging, genome organization, and distribution of specific repeated DNA sequences for homologous chromosome cognition and pairing, and the distribution of recombination events along the chromosomes. 相似文献
20.
Iodinated ribosomal RNA was hybridized to human metaphase chromosomes in a test of the effectiveness of iodinated products for in situ hybridization studies in a diploid system. The results indicate that 125I is a feasible alternative as a source of radioactivity for autoradiographic mapping studies. 相似文献