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At least 19 recordsLinked to original sources
PubMed2001

Telomere associations in interphase nuclei: possible role in maintenance of interphase chromosome topology.

The relative sizes of individual telomeres in cultured human cells under conditions of cell cycling, replicative quiescence, cell transformation and immortalization were determined using quantitative fluorescence in situ hybridization (Q-FISH) with a telomere-specific peptide nucleic acid (PNA) probe. Results obtained from analysis of telomere length profiles (TLPs), which display the distribution of relative telomere lengths for individual cells, confirmed telomere length heterogeneity at the single cell level and proportional shortening of telomere length during replicative aging of virus-transformed cells. TLPs also revealed that some telomeric ends of chromosomes are so closely juxtaposed within interphase nuclei that their fluorescent signals appear as a single spot. These telomeric associations (TAs) were far more prevalent in interphase nuclei of noncycling normal and virus-transformed cells than in their cycling counterparts. The number of interphase TAs per nucleus observed in late-passage E6/E7-transformed cells did not increase during progression to crisis, suggesting that telomere shortening does not increase the frequency of interphase TAs. Furthermore, interphase TAs were rarely observed in rapidly cycling, telomerase-positive, immortalized cells that exhibit somewhat shortened, but stabilized, telomere length through the activity of telomerase. Our overall results suggest that the number of interphase TAs is dependent more on whether or not cells are cycling than on telomere length, with TAs being most prominent in the nuclei of replicatively quiescent cells in which nonrandom (even preferred) chromosome spatial arrangements have been observed. We propose that interphase TAs may play a role in the generation and/or maintenance of nuclear architecture and chromosome positional stability in interphase nuclei, especially in cells with a prolonged G(1)/G(0) phase and possibly in terminally differentiated cells.

Cell Cycle↗
PubMed1997

Experimental induction of prenucleolar bodies (PNBs) in interphase cells: interphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells.

Recently, it was shown that a short exposure of living mammalian cells to low ionic strength buffers (hypotonic shock) caused partial or almost complete unraveling of interphase nucleoli. However, when the cells were released from the hypotonic shock and transferred to normal isotonic medium, functionally active and structurally integral nucleoli were reassembled at their initial positions within interphase nuclei. Here, we show further that this process is accompanied by the appearance of numerous discrete extranucleolar bodies, which have striking similarities to the prenucleolar bodies (PNBs) observed in untreated cells at telophase of mitosis. (1) Like PNBs at mitosis, hypotonically induced interphase PNBs are composed of RNA-positive granules and fibrils, contain the major nucleolar protein B23 and silver-binding proteins, but lack DNA and RNA polymerase I transcription factor UBF. (2) As for mitotic PNBs, disappearance of the interphase PNB counterparts coincides with the increase in size of reconstructed nucleoli. (3) Addition of actinomycin D does not prevent assembly of interphase PNBs, but does arrest their coalescence with the chromosomal nucleolus-organizing regions and blocks the complete reformation of nucleoli. It is concluded that the assembly of PNBs generally observed at telophase of mitosis can be induced experimentally in nuclei of interphase mammalian cells in vivo. At interphase, this process is probably initiated by changes in the intracellular ionic environment.

Animals↗
PubMed2004

Proteomics analysis of the centromere complex from HeLa interphase cells: UV-damaged DNA binding protein 1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently co-localized with the centromeric region in interphase.

CENP-A, a centromere-specific histone H3, is conserved throughout eukaryotes, and formation of CENP-A chromatin defines the active centromere region. Here, we report the isolation of CENP-A chromatin from HeLa interphase nuclei by chromatin immunoprecipitation using anti-CENP-A monoclonal antibody, and systematic identification of its components by mass spectrometric analyses. The isolated chromatin contained CENP-B, CENP-C, CENP-H, CENP-I/hMis 6 and hMis 12 as well as CENP-A, suggesting that the isolated chromatin may represent the centromere complex (CEN-complex). Mass spectrometric analyses of the CEN-complex identified approximately 40 proteins, including the previously reported centromere proteins and the proteins of unknown function. In addition, we unexpectedly identified a series of proteins previously reported to be related to functions other than chromosome segregation, such as uvDDB-1, XAP8, hSNF2H, FACTp180, FACTp80/SSRP1, polycomb group proteins (BMI-1, RING1, RNF2, HPC3 and PHP2), KNL5 and racGAP. We found that uvDDB-1 was actually localized to the centromeric region throughout cell cycle, while BMI-1 was transiently co-localized with the centromeres in interphase. These results give us new insights into the architecture, dynamics and function of centromeric chromatin in interphase nuclei, which might reflect regulation of cell proliferation and differentiation.

Autoantigens↗
PubMedDate not supplied

[The interphase death of dividing cells. The effect of oxygen and the pH of the medium on the interphase death of Ehrlich ascites carcinoma cells under the action of ionizing radiation].

Interphase death of in vitro irradiated (200 Gy) Ehrlich ascites tumor cells was studied as a function of oxygenation level and medium pH. The presence of oxygen both during and after irradiation as well as the increase in pH from 7.4 to 8.1 were shown to increase interphase death rate. The pronounced threshold dependence of interphase death of cells upon their concentration may be attributed to hypoxia occurring in a pericellular medium when cells concentration exceeds the threshold.

Animals↗
PubMed1996

Metaphase and interphase mapping by FISH: improvement of chromosome banding and signal resolution in interphase nuclei by means of iterative deconvolution.

FISH images obtained with conventional epifluorescence microscopes are always blurred by glare and out of focus light emissions. In order to restore high contrast images, a procedure based on a modelling of the optical system in the microscope was developed and used for the processing of images acquired with a cooled CCD camera mounted on a fluorescence microscope. This procedure was tested on images of both mouse and human chromosomes stained with DAP1 and on images of interphase nuclei hybridized with pairs of cosmid probes. This method improves the definition and the sharpness of the DAPI G-banding and thus facilitates and speeds up the identification of chromosomes. When performed on images of interphase cell nuclei, this procedure allows the discrimination of fluorescent signals which appear partially overlapping on raw images. This significant improvement of spatial resolution is of particular interest for ordering sets of probes on DNA fibers.

Animals↗
PubMedDate not supplied

[The interphase death of dividing cells. The relation of interphase death to cell energy requirements].

In experiments with irradiated cells of Chinese hamster and Ehrlich ascites tumor a study was made of the influence of energy provision on their interphase death rate. The presence of the uncoupler of respiration and oxidative phosphorylation--carbonyl cyanide-3-chlorophenylhydrazone--in a medium without glucose was shown to drastically increase the interphase death rate of cells of both types, whereas this effect was not observed in a medium with glucose.

Animals↗
PubMed1994

Interphase cytogenetics as an adjunct in the cytodiagnosis of urinary bladder carcinoma. A comparative study of cytology, flow cytometry and interphase cytogenetics in bladder washes.

To determine the role of interphase cytogenetics as an adjunct in the cytodiagnosis of transitional cell carcinoma (TCC) in bladder washes, 40 separate samples were prospectively evaluated by conventional cytology, flow cytometry (FCM) and interphase cytogenetics using fluorescence in situ hybridization (FISH) to detect numerical chromosomal abnormalities involving chromosomes 8 and 12. The cases, all of which had subsequent histologic confirmation, were composed of 26 transitional cell carcinomas and 14 benign lesions. Cytology, FCM and FISH were concordant in 19 of 31 (61%) instances. The false-negative rates of the three parameters were as follows: cytology, 38.5%; FCM, 28.5%; FISH, 27%. The false-positive rates were 0%, 20% and 0%, respectively. The relatively lower sensitivity of cytology is attributed to sampling problems since 7 of the 10 false-negative cases were histologically high grade (grades 2 and 3) and should have been detected easily by conventional cytology if tumor cells were present. Thus, the sensitivity and specificity of FISH in detecting malignant cells of TCC in bladder washes is comparable to that of conventional cytology and FCM. When, as in the present study, there are insufficient numbers of cells for FCM analysis, the detection of numerical chromosomal abnormalities by FISH studies may serve as an adjunct in the diagnosis of TCC in bladder washes.

Carcinoma, Transitional Cell↗
PubMed1976

Laser UV microirradiation of interphase nuclei and post-treatment with caffeine. A new approach to establish the arrangement of interphase chromosomes.

Laser UV microirradiation of Chinese hamster interphase cells combined with caffeine post-treatment produced different patterns of chromosome damage in mitosis following irradiation of a small area of the nucleus that may be classified in three categories: I)intact metaphase figures, II)chromosome damage confined to a small area of the metaphase spread, III)mitotic figures with damage on all chromosomes. Category III might be the consequence of a non-localized distortion of nuclear metabolism. By contrast, category II may reflect localized DNA damage induced by microirradiation, which could not be efficiently repaired due to the effect of caffeine. If this interpretation is right, in metaphase figures of category II chromosome damage should occur only at the irradiation site. The effect might then be used to investigate neighbourhood relationships of individual chromosomes in the interphase nucleus.

Animals↗
PubMed1994

Interphase cytogenetics of melanocytic neoplasms: numerical aberrations of chromosomes can be detected in interphase nuclei using centromeric DNA probes.

This study shows that fluorescence in situ hybridization (FISH) to thin sections cut from paraffin-embedded material can be used to distinguish between groups of melanocytic neoplasms and thus may be useful as an investigational and diagnostic tool. FISH with a probe for a repeated, alpha satellite sequence specific to chromosome 17 was used to investigate the chromosomal composition of dysplastic (or Clark's nevus) and Spitz's nevi and malignant melanomas. Hybridization was to thin (approximately 6 microns) sections cut from paraffin blocks. The number of signals per nucleus in normal diploid cells is expected to be less than 2 since the sections are thinner than one nuclear diameter. Keratinocytes and lymphocytes in these same sections showed 1-2 signals per nucleus with a mean of 1.2. Dysplastic nevi showed 1-4 hybridization signals per nucleus with a mean of 1.5. Spitz's nevi showed 1-2 signals per nucleus with a mean of 1.3. Melanomas showed 1-6 signals per nucleus with a mean of 2.1. We were thus able to use FISH to demonstrate differences in chromosome numbers between groups of benign and malignant melanocytic neoplasms. Technical improvements in the near future can be expected to result in more precise estimates of chromosomal number.

Centromere↗
PubMedDate not supplied

[The intercellular interactions in the interphase death of irradiated thymocytes. A study of the influence of different cells on thymocyte interphase death].

The influence of different cells on death and pycnosis of thymocyte nuclei after in vitro irradiation has been investigated. It has been shown that the removal from the thymocyte suspension of cells, having the activity of natural killers, medullar thymocytes, and macrophages, does not influence the radiation-induced damage to cortical thymocytes. The injury of exposed thymocytes decreases, however, after incubation them with nonirradiated thymocytes or cultured cells, the efficiency of the latter being dependent on the type of cells. The data obtained may indicate that the exposed thymocytes interact with each other and exchange some factors that promote their injury.

Animals↗
PubMed1993

Ionizing radiation induces two forms of interphase chromosome breaks in Chinese hamster ovary cells that rejoin with different kinetics and show different sensitivity to treatment in hypertonic medium or beta-araA.

We have shown previously that incubation of irradiated plateau-phase CHO cells in hypertonic growth medium during the period normally allowed for chromosome condensation, in the premature chromosome condensation (PCC) assay, uncovers a form of interphase chromosome breaks that rejoin with fast kinetics (t1/2 = 1.5 min). Here, we report that incubation with beta-arabinofuranosyladenine (beta-araA), an inhibitor of DNA, chromosome, and cellular repair processes, during the same period uncovers a different form of interphase chromosome breaks that rejoin with slower kinetics (t1/2 = longer than 15-20 min). The yield of interphase chromosome breaks increased from 2.0 breaks/cell/Gy in untreated control cells to 3.6 breaks/cell/Gy in cells exposed to 1 mM beta-araA, and was the same as that observed in cells treated in hypertonic medium (500 mM NaCl). Simultaneous exposure to beta-araA and hypertonic medium increased the yield of interphase chromosome breaks further to 5.3 breaks/cell/Gy. This increase was consistent with an additive effect of each treatment on the overall yield of breaks, and suggested that hypertonic medium and beta-araA affect distinct and independent subsets of radiation-induced interphase chromosome breaks. We tested further the notion of independence by measuring rejoining of interphase chromosome breaks sensitive to hypertonic treatment in the presence of 1 mM beta-araA, and vice versa, rejoining of interphase chromosome breaks sensitive to beta-araA during and after treatment in hypertonic medium (500 mM NaCl, 20 min); under both sets of conditions each treatment caused maximal expression of prematurely condensed chromosome breaks responding sensitively to it when given immediately after irradiation. There was no change in the rejoining kinetics of interphase chromosome breaks sensitive to hypertonic treatment in the presence of beta-araA, and no change in the rejoining kinetics of interphase chromosome breaks sensitive to beta-arA in cells treated in hypertonic medium. These results are consistent with the hypothesis that exposure to ionizing radiation leads to the induction of two forms of prematurely condensed chromosome breaks that can be distinguished from each other on the basis of their repair kinetics and their differential sensitivity to treatment with beta-araA or hypertonic medium. In direct analogy to a classification proposed previously for potentially lethal damage (PLD) based on a similar set of experiments, we introduce the terms alpha form and beta form of interphase chromosome breaks for the slow, beta-araA-sensitive, and the fast, hypertonic treatment-sensitive form, respectively. We also propose that there is a correlation between alpha and beta form of interphase chromosome breaks and alpha and beta form of PLD, and present evidence suggesting that fast and slowly repairing DNA double-strand breaks underlie fast and slowly repairing interphase chromosome breaks.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Evidence that the product of the xrs gene is predominantly involved in the repair of a subset of radiation-induced interphase chromosome breaks rejoining with fast kinetics.

We classified interphase chromosome breaks into alpha and beta forms to study the requirement for the xrs gene product in the repair of each of these forms of damage. The alpha form of damage comprises radiation-induced interphase chromosome breaks whose rejoining is slow and sensitive to treatment with beta-arabinofuranosyladenine (beta-araA), whereas the beta form of damage comprises interphase chromosome breaks whose rejoining is fast and sensitive to treatment in hypertonic medium. Interphase chromosome breaks of the alpha form are visualized in plateau-phase cells by premature chromosome condensation (PCC) carried out in the absence of any treatment during the condensation period. More interphase chromosome breaks of the alpha form can be uncovered by treatment with beta-araA during the period of PCC. Interphase chromosome breaks of the beta form are not visualized in experiments using standard PCC protocols but can be uncovered by treatment in hypertonic growth medium during the period allowed for PCC. In the present report, we show that the yield of interphase chromosome breaks of the alpha form is similar in CHO and xrs-5 cells and demonstrate that xrs-5 cells rejoin this type of interphase chromosome breaks with an efficiency similar to that observed in repair-proficient CHO cells. Furthermore, we provide evidence supporting the notion that xrs-5 cells are deficient in the rejoining of the beta form of interphase chromosome breaks. These results strongly suggest that the product of the xrs gene is required predominantly in the repair of the beta form of interphase chromosome damage and emphasize the need for discrimination between different forms of interphase chromosome breaks in irradiated cells.

Animals↗
PubMed1996

Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes.

The chromatin in interphase nuclei is much less condensed than are metaphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuclei than on metaphase chromosomes. In mammalian species it has been demonstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance between the two probes. The interphase mapping strategy has never been applied in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) camera system, we demonstrate that DNA probes in the 4 to 8 kb range can be detected on both metaphase and interphase chromosomes in maize. DNA probes pA1-Lc and pSh2.5.SstISalI, which contain the maize loci a1 and sh2, respectively, and are separated by 140 kb, completely overlapped on metaphase chromosomes. However, when the two probes were mapped in interphase nuclei, the FISH signals were well separated from each other in 86% of the FISH sites analyzed. The average interphase distance between the two probes was 0.50 micron. This result suggests that the resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of another pair of probes, ksu3/4 and ksu16, which span the Rp1 complex controlling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped genetically approximately 4 cM apart and their FISH signals were also overlapped on metaphase chromosomes. These two probes were separated by an average of 2.32 microns in interphase nuclei. The possibility of estimating the linear DNA distance between ksu3/4 and ksu16 is discussed.

Cell Nucleus↗
PubMed1991

Chromosome topology in mammalian interphase nuclei.

Since 1968, when Comings published the pioneering paper on "the rationale for an ordered arrangement of chromatin in the interphase nucleus," technical methods have progressed tremendously and improved our understanding of interphase organization. The existence of highly ordered organizational patterns of the cell nucleus appears to be beyond any doubt and it is difficult to escape the conclusion that interphase chromosome topology is important for the complex regulation of the many varied and interrelated nuclear processes. However, it is worth emphasizing that a universally valid principle of chromosome arrangement does not exist and, therefore, any generalization of interphase patterns can be misleading. The factors of order according to which the chromosomes are arranged inside the nucleus are manifold: (1) Individual chromosomes remain in spatially separated domains throughout interphase, preventing an intermingling of the decondensed euchromatin. (2) Chromosome regions that contain constitutive heterochromatin associate into larger chromocenters. (3) In most cell types direct associations between interphase domains of homologous chromosomes are not observed. In others homologous heterochromatic regions tend to be paired preferentially. (4) Interphase chromosomes do not float freely in the nucleoplasm; they are associated to varying degrees with the nuclear membrane and other components of the nuclear scaffold. The number of attachment sites for each chromosome to the nuclear membrane is relatively low. (5) The positions of centromeres (and pericentromeric heterochromatin) are nonrandom and characteristic of each cell type. Specific centromere movements occur during the cell cycle, during differentiation, and under certain pathophysiological conditions. (6) The telomeric chromosome ends are particularly prone to associate in certain somatic cell types and in meiotic prophase cells. (7) The arrangement of repetitive DNA families appears to determine a structural framework of the interphase nucleus. Different cell types of one organism can exhibit marked differences in their repetitive DNA framework, whereas cells that are in an identical differentiated state or an identical phase of the cell cycle often show comparable interphase patterns even in evolutionarily distant species. (8) The various steps of ribosome biogenesis take place in a precise fashion within a separate nuclear domain, the nucleolus. The topologically well-defined nucleolar substructures are required for rDNA transcription and pre-rRNA processing. (9) A compartmentalization of transcriptional and processing events is also evident in the rest of the nucleus. However, it is not yet known if the in situ sites of transcription and RNA processing for a particular (nonribosomal) gene or gene family are actually adjacent. (10) DNA replication is precisely spatiotemporally regulated within the nucleus. The replication domains are immobilized on the nuclear matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Treatment of cells with alkaline borate buffer extends the capability of interphase FISH mapping.

Interphase fluorescence in situ hybridization (FISH) has been shown to be a means to map DNA sequences relative to each other in the 100 kb to 1-2 Mb genomic-separation range. At distances below 0.1 Mb, probe sites are infrequently resolved in interphase chromatin. In the 0.1- to 1-Mb range, interphase chromatin can be modeled as a freely flexible chain. The mean square interphase distance between two probes is proportional to the genomic separation between the probes on the linear DNA molecule. Above 1-2 Mb, the relationship between interphase distance and genomic separation changes abruptly and appears to level off. We have used alkaline-borate treatment to expand the capability of interphase FISH mapping. We show here that alkaline-borate treatment increases nuclear diameter, the interphase distance between probes on homologous chromosomes, and the distance between probes on the same chromosome. We also show that the mean square distance between hybridization sites in borate-treated nuclei is proportional to genomic separation up to 4 Mb. Thus, alkaline-borate treatment enhances the capability of interphase FISH mapping by increasing the absolute distance between probes and extending the range of the simple relationship between interphase distance and genomic separation.

Borates↗