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PubMed1991

Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei.

We have used the proximity of probe hybridization sites in interphase chromatin to derive the order of DNA sequences in a 2-3-Mbp region of human chromosome Xq28. The map generated bridges the results of genetic and pulsed-field gel electrophoresis mapping to produce a more complete map of Xq28 than possible with either of these other techniques alone. Two-color fluorescence in situ hybridization (FISH) was used to detect the positions of two or more probes in G1 male interphase nuclei. We show that cosmids that are 50 kbp to 2-3 Mbp apart can be ordered rapidly with two alternative approaches: (1) by comparing the average measured distance between two probes and (2) simply by scoring the order of red and green fluorescent dots after detection of three or more probes with two fluorochromes. The validity of these approaches is demonstrated using five cosmids from a region spanning approximately 800 kbp that includes the factor VIII (F8), glucose-6-phosphate dehydrogenase (G6PD), and color-vision pigment (CV) genes. The cosmid map derived from interphase mapping is consistent with the map determined by restriction-fragment analysis. The two interphase mapping approaches were then used (1) to orient the F8/CV cluster relative to two markers, c1A1 and st14c, which we show by metaphase mapping to be proximal to the F8/CV cluster, (2) to position st14c (DXS52) between c1A1 and F8, and (3) to orient the CV gene cluster relative to G6PD by using two CV-flanking cosmids, 18b41 and fr7. The probe order in Xq28 derived from interphase proximity is cen-c1A1-st14c-5'F8 (p624-p542-p625)-G6PD-18b41-3' green-green-red-fr7-tel. We also show that, to determine their order by using metaphase chromosomes, sequences must be at least 1 Mbp apart, an order of magnitude greater than required in interphase chromatin. The data show that FISH mapping is a simple way to order sequences separated by greater than or equal to 50 kbp for the construction of long-range maps of mammalian genomes.

Chromosome Mapping↗
PubMed1990

Detection of monosomy 7 in interphase cells of patients with myeloid disorders.

Six patients, five with acute myeloid leukemia (AML) and one with a myelodysplastic syndrome (MDS), were found to have monosomy 7 by conventional cytogenetics at diagnosis. Repetitive DNA sequences from the heterochromatic region of human chromosomes 1 and 7 were used as probes for in situ hybridization experiments on interphase cells of these patients. A double hybridization protocol was used to reveal the particular chromosomes as distinct spots or clusters of signals within interphase nuclei. The chromosome 1 sequence served as an internal control. Simultaneous detection of the sequences showed the presence of two normal number 1 chromosomes and a missing 7 chromosome from individual cells. While cytogenetic preparations showed only -7 metaphases in 3 AML and 1 MDS patients, in situ hybridization of interphase cells showed many normal cells as well as the presence of -7 in fully mature granulocytes. One AML patient studied in remission showed only normal metaphases yet had 9% interphase cells with a missing 7 and relapsed within 3 months. We conclude that examination of interphase cells by in situ hybridization provides clinically useful data since every cell including mature granulocytes can be examined, the lineage of a cell can be determined, and efficacy of differentiation therapy can be evaluated.

Chromosome Deletion↗
PubMed1994

Microtubule converging centers and reorganization of the interphase cytoskeleton and the mitotic spindle in higher plant Haemanthus.

We analyzed the distribution and orientation of transitory microtubule structures, microtubule converging centers, during interphase and mitosis in endosperm of the higher plant Haemanthus. In interphase the pointed tips of microtubule converging centers are associated with the nuclear envelope. Their orientation gradually reverses during prophase, and the tips tend to point away from the nucleus. From prometaphase through early telophase, microtubule converging centers are present predominantly in the cytoplasm at the polar region. They are either "free" or associated with chromosomes or microtubule bundles. In late telophase, pointed tips of microtubule converging centers are again associated with the reconstructed nuclear envelope and, additionally, they often appear in the phragmoplast area. The orientation of microtubule converging centers seems to be directly correlated to the previously determined microtubule polarity, with the converging tip being minus and the diverging one, plus. Elevated temperature (35 degrees-37 degrees C) enhances the number of microtubule converging centers in the cytoplasm and at the nuclear envelope. This is especially pronounced during the telophase-interphase transition and in some interphase cells, indicating temperature and stage dependence. Our data imply that microtubule converging centers bind together MT minus ends and, thus, control the predominant direction of elongation and shortening of microtubule arrays. We argue that these configurations are instrumental during the reorganization of interphase cytoskeleton and mitotic spindle in Haemanthus endosperm.

Cell Cycle↗
PubMed1995

Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro.

Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithelial, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like-structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.

Animals↗
PubMed1995

Prenatal diagnosis of trisomy 21 using interphase fluorescence in situ hybridization of post-replicated cells with site-specific cosmid and cosmid contig probes.

Interphase fluorescence in situ hybridization (FISH) with chromosome 21-specific cosmid clones was used to identify trisomy 21 in cultured and uncultured amniotic cells. Two novel site-specific cosmid clones (regions 21q22 and 21qtel) were compared with a cosmid contig (Zheng et al., 1992). Correct identification of chromosome 21 copy number was made in 65-75 per cent of trisomic cells and in 70-75 per cent of normal disomic cells by using all the tested probes. However, the chromosome 21-specific telomeric probe (cos 17F8) showed the best results due to more intense and clearly visible hybridization. Utilization of a directly fluorophorated telomeric probe using Cy3-dCTP and FluorX-dCTP allows accurate detection of chromosome 21 in a fast 'one-step' FISH procedure on uncultured interphase nuclei. In addition, we compared the efficacy of FISH analysis for the total population of interphase cells and cells in the post-replication (late S, G2) periods of the cell cycle. Selective scoring of cells in the post-replicative period (showing a pair of hybridization signals on each chromatid of the replicated interphase chromosome) increased the number of informative nuclei by up to 95-97 per cent. This approach allows cells with overlapping chromosomes, artificial double hybridization signals on separate chromatids in interphase chromosomes, background hybridization, and polyploid cells to be analysed. Application of directly labelled telomeric cosmid probes and integral analysis of hybridized nuclei in the pre- and post-replication periods of the cell cycle may help to further improve the prenatal detection of trisomy 21.

Amniotic Fluid↗
PubMed1994

Interphase AgNOR quantity is not related to DNA content in 11 established human cancer cell lines.

The relationship between interphase AgNOR quantity and DNA ploidy was evaluated in 11 cell lines derived from human tumors of different origin. Interphase AgNOR quantity was measured by morphometric image analysis and DNA content by cytofluorometry. The mean interphase AgNOR area ranged from 5.56 to 21.12 microns 2 and DNA ploidy from 2.05 to 3.30c. The interphase AgNOR value and DNA content were not significantly related (r = 0.23, P = 0.52). No significant correlation was found between DNA content and cell doubling time, whereas a strict relationship was confirmed to exist between interphase AgNOR quantity and rapidity of cell proliferation.

Cell Division↗
PubMed1994

Effect of telomeres on the interphase location of adjacent regions of the human X chromosome.

Chromosomes occupy specific nonrandom domains in the interphase nucleus of eukaryotic cells. We have used a Chinese hamster-human somatic cell hybrid line containing a single human X chromosome to study the interphase distribution of the Xp telomere using fluorescent in situ hybridization and optical sectioning. A derivative cell line in which the X chromosome has been broken at Xq22-24 and healed by the addition of cloned human telomeric sequences was also studied to determine if introduction of these sequences at a previously interstitial site changed its location in interphase. The endogenous Xp telomere occupies a specific, nonrandom, internal domain. Introduction of a telomere at a previously interstitial site did not alter the interphase nuclear location of that site. The results suggest that nonrandom interphase location of telomeres may not be determined solely by the DNA sequence of the telomere.

Cell Nucleus↗
PubMed1994

In vitro investigations of interphase and metaphase argyrophilic nucleolar organizer regions and cellular proliferation in the human urothelial cancer cell line HOK-1.

Detailed investigation of cell growth and nucleolar organizer region associated argyrophilic proteins (Ag-NORs) is necessary to assess a possible impact of Ag-NOR quantification on the diagnosis and prognosis of tumours. In this study, cellular proliferation of the transitional-cell carcinoma cell line HOK-1 was modulated over a period of 11 days by starvation and subsequent medium addition. Proliferation was determined daily by DNA flow cytometric estimation of S-phase fraction (SPF) and mitotic index (MI) calculation. The number and area of interphase Ag-NORs were quantified by automated image analysis daily and the number of Ag-NOR bearing chromosomes in metaphase was counted. In interphase nuclei, Ag-NOR area showed a highly significant correlation with SPF (p < 0.0001) whereas interphase Ag-NOR number showed significant correlation with MI (p < 0.05). A positive relationship between the number of Ag-NOR bearing chromosomes in metaphases and cellular proliferation was also observed. There is variability in Ag-NOR quantity during interphase and metaphase depending on growth conditions in vitro. Correlations of the number of interphase Ag-NORs with the MI on one hand and Ag-NOR area with SPF on the other provide further evidence that distribution and quantity of Ag-NORs are strongly influenced by the cell cycle phase within the structural-functional unit of the nucleolus.

Carcinoma, Transitional Cell↗
PubMed1985

Individual interphase chromosome domains revealed by in situ hybridization.

The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 micron wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.

Animals↗
PubMed1987

The blocked pinocytic activity of mitotic cells is restored in mitotic-interphase hybrids.

During mitosis there is an abrupt inhibition of a wide range of membrane functions, including fluid-phase and adsorptive pinocytosis. We have used cell hybrids formed between mitotic and interphase cells to approach the mechanism of this inhibition. We report that fluid pinocytosis is reactivated in the mitotic partner of hybrids formed between mitotic and interphase Chinese hamster ovary (CHO) cells. It thus appears that the interphase cell provides some necessary element(s) for membrane activity during mitosis. This dominance of interphase membrane properties stands in contrast with earlier evidence that mitotic nuclear properties dominate in similar mitotic-interphase hybrids.

Animals↗
PubMed1992

Longitudinal cytogenetic study of metaphase and interphase cells in childhood monosomy 7 syndrome.

A 15-year-old male with myelodysplastic syndrome (MDS) characterized by monosomy 7 was cytogenetically evaluated by metaphase karyotyping and fluorescence in situ hybridization (FISH) of interphase cells at six different points during the course of his disease. At diagnosis, there was complete agreement between metaphase and interphase findings. Interphase analysis alone provided important cytogenetic information on the first specimens received following intensive combination chemotherapy and bone marrow transplantation where metaphase analyses were uninformative. The detection of a minor post-treatment monosomy 7 population by interphase but not metaphase studies may have identified minimal residual disease prior to recurrence of MDS. From this longitudinal study, it is concluded that metaphase and interphase cytogenetic analyses form complementary approaches and that use of both provides greater analytical power when appropriate chromosome markers are available.

Adolescent↗
PubMed1993

Interphase FISH and morphologic analysis of AML.

Interphase cytogenetic analysis of peripheral white blood cells from a patient with acute myelogenous leukemia (AML) who was previously diagnosed with trisomy 8 was performed for the purpose of documenting clonal response to intensive chemotherapy. DNA in situ hybridization was performed using a probe specific for chromosome 8 alpha-satellite DNA sequences. Cells were stained with Wright stain prior to interphase analysis and photographed to allow correlation of cell morphology with abnormal karyotype. Prior to chemotherapy, the patient's leukocyte differential contained 8% blasts; interphase analysis revealed 23% trisomy 8 cells, including mature granulocytes. Forty days after the start of chemotherapy, at which point the patient had attained clinical remission, interphase analysis revealed only 2% cells with three signals, which was not statistically significant when compared with our own series of controls. The use of interphase FISH analysis in this case provides additional evidence that some leukemic blasts may be capable of limited differentiation in vivo and also suggests a differential sensitivity to chemotherapy between cytogenetically normal and abnormal hematopoietic precursor cells.

Aged↗
PubMed1998

Detection of structural chromosome damage in rat interphase cells using region-specific fluorescence in situ hybridization probes developed by microdissection.

Cytogenetic analysis using fluorescence in situ hybridization (FISH) was employed to detect structural chromosome aberrations in interphase. We generated DNA probes specific for rat chromosome regions 1q11-12, 1q31-35 and 1q51-53 by microdissection and degenerate oligonucleotide-primed PCR. Targeted regions were labeled in unique colors by FISH. Abnormal cells were identified on the basis of alterations in the physical distance between the hybridization signals. To evaluate the ability of these probes to quantify chromosome aberrations in interphase, rats were acutely exposed whole-body to 0, 1, 2, 3 or 4 Gy of 137Cs gamma rays. Eight days later, peripheral blood, bone marrow, lung and pancreas were removed and hybridized with the probes. Multi-color FISH analysis showed dose-responsive frequencies of abnormal interphase nuclei in peripheral blood and bone marrow cells. In lung and pancreas, on the contrary, no increase in the frequency of the abnormal interphases was observed. However, chromosome damage was observed when primary lung cells, obtained from rats irradiated 8 days previously, were cultured for three days. Detection of rearranged signals after in vitro tissue culture was attributed to the movement of chromosome domains that accompanies mitosis. The use of region-specific painting probes appears useful for detecting structural chromosome damage in interphase cells of rat tissues, although further optimization is still needed to improve the method.

Animals↗
PubMedDate not supplied

Identification of triploidy by DA/DAPI staining of trophoblastic interphase nuclei.

Combined staining of non-cultured interphase nuclei from hydatidiform moles with distamycin A and DAPI resulted in the presence of specific stained interphase bodies. A mean number of six interphase bodies was observed in moles with a diploid karyotype and a mean number of nine interphase bodies was observed in triploid moles. It is concluded that the interphase bodies are primarily due to specific staining of the large heterochromatic areas on chromosomes 1, 9 and 16. The method may permit a rapid measurement of the ploidy of non-cultured hydatidiform moles and identify partial, triploid moles.

Cell Nucleus↗
PubMed1992

Variations in alphoid DNA sequences escape detection of aneuploidy at interphase by FISH technique.

The advent of a new staining technique, termed fluorescence in situ hybridization (FISH), allows the rapid identification of the genomic constitution of an individual with aneuploidy even in interphase nuclei through the use of a series of chromosome-specific DNA probes, an approach termed "interphase cytogenetics." However, alphoid DNA sequences of every centromere are polymorphic (heteromorphic), and the number of targeted sequences may be below the detection level of a specific DNA probe, thus escaping detection and resulting in the imprecise identification of the chromosomal constitution at interphase. The limitations associated with the FISH technique have dire consequences which are emphasized here with an example in which the presence of an additional chromosome 21 in two siblings born consecutively with trisomy 21 (Down syndrome) was not detected by "interphase cytogenetics." The copy number of alphoid DNA sequences of one of the paternal chromosomes 21 was low and resulted in discordance between domain numbers at interphase and actual chromosome numbers at metaphase in both children. This is an isolated incident that could have led to a misdiagnosis if FISH were the only test employed. Although the advantages of this technology are undeniably enormous, the present finding has made it apparent that precise standards and reliability of the procedure must be established prior to its routine application.

DNA Probes↗
PubMed1998

Protein kinase inhibitors induce the interphase transition by inactivating mitogen-activated protein kinase in mouse eggs.

The role of mitogen-activated protein (MAP) kinase in mouse egg activation induced by protein kinase inhibitors and a protein tyrosine kinase (PTK) inhibitor was investigated. Separated egg proteins were first probed with anti-Active MAP kinase antibody and then re-probed with anti-ERK2 antibody. Staurosporine and Ro-31-8220, at concentrations that normally inhibit protein kinase C, did not affect egg activation or MAP kinase activity, while higher dosages caused egg activation. Staurosporine at 2 microM induced the metaphase-interphase transition without emission of the second polar body (PB2), while Ro-31-8220 at 40 microM induced PB2 emission, first cleavage, and then the transition to interphase. Half the eggs were also activated by the PTK inhibitor genistein. In each treatment, the proportion of eggs that entered interphase was well correlated with the degree of MAP kinase inactivation. Artificial activation of this kinase by okadaic acid overcame the interphase transition. These data suggest that protein kinase inhibitors and a protein tyrosine kinase inhibitor induce the interphase transition by inactivating MAP kinase in mouse eggs.

Animals↗
PubMed1998

Non-Rabl patterns of centromere and telomere distribution in the interphase nuclei of plant cells.

At the anaphase of cell divisions, the divided chromosomes move to the two poles, with the centromeres as heads and telomeres as tails. Such a polarized orientation of centromeres and telomeres is believed to be preserved in the interphase and is known as Rabl model. We analyzed the distributions of centromeres and telomeres in interphase nuclei from several plant species. Although Rabl polarity was observed in wheat, rye, barley and oats, non-Rabl patterns were discovered in sorghum, rice and maize. In the non-Rabl patterns, both centromeres and telomeres were dispersed throughout the interphase nucleus, except in the area occupied by the nucleolus. Both Rabl and non-Rabl distribution patterns of centromeres and telomeres were consistent in interphase nuclei derived from meristematic root tip cells, microspore mother cells and differentiated leaf cells. Our study demonstrated that there is a diversity of interphase chromatin organization and that the classical Rabl model is not universal in plant species.

Avena↗
PubMed1997

Regional differences in the compaction of chromatin in human G0/G1 interphase nuclei.

The large-scale structure of chromatin corresponding to G- and R-bands in human G0/G1 interphase nuclei was compared. Fluorescence in situ hybridization (FISH) was used to measure the interphase distance between 42 pairs of probes separated by 0.1-1.5 Mbp. The probe pairs were derived from 21q22.2 and Xp21.3, G-band positive regions, and from 4p16.3, 6p21.3, and Xq28, R-band positive regions. Distributions of measured interphase distances in all regions approximated a Rayleigh distribution, suggesting that the chromatin follows a random-walk path over this range. A linear correlation of mean-square interphase distance and genomic separation, also indicative of random-walk folding, was observed in all regions. The slope of the correlation observed using probes from G-band regions was systematically lower than that from R-band regions. The difference in the slope between Xp21.3 and Xq28 was particularly striking and was observed in normal fibroblast cells, fixed alternatively with methanol and acetic acid or paraformaldehyde, and HeLa cells. These results demonstrate regional differences in large-scale chromosome structure during interphase, with the more openly configured chromatin corresponding to R-bands.

Chromatin↗