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PubMed1999

Dynamics of structure-function relationships in interphase nuclei.

The interphase nucleus is a topologically ordered, three-dimensional structure. While it remains unclear whether this structural organization also represents compartmentalization of function, the presence of the latter would likely be reflected in the spatial coupling of molecular factors involved in related events. This review summarizes morphological evidence, derived from in situ experiments, which indicates the existence of compartmentalization of both chromatin and non-chromatin components in the interphase nucleus. Moreover, the review addresses the spatial relationships of these components relative to each other and correlates these spatial relationships with such nuclear functions as transcription, splicing and nucleo-cytoplasmic transport of pre-mRNA. Given that it is increasingly recognized that such spatial relationships are dynamic, the review also addresses the emerging concept that the spatial intranuclear organization changes with changes in cell function, a concept which supports the hypothesis that the spatial organization of the interphase nucleus may represent one of the fundamental control mechanisms in gene expression.

Animals↗
PubMed1998

Prenatal detection of aneuploidy by directly labeled multicolored probes and interphase fluorescence in situ hybridization.

OBJECTIVE: To detect aneuploidy of chromosomes 13, 18, 21, X, and Y with use of new, directly labeled, multicolored, commercially available DNA probes from interphase cells of amniotic fluid (AF). MATERIAL AND METHODS: The hybridization sites of the five probes were validated by metaphase analysis. The fluorescence in situ hybridization (FISH) normal range was determined from a series of normal AF specimens and tested on a series of normal and abnormal specimens. RESULTS: The hybridization efficiencies of the five probes were 100%. The mean AF interphase disomic signal patterns for chromosomes 13, 18, 21, XX, and XY were 95.9%, 89.1%, 94.3%, 94.7%, and 98.7%, respectively. Of a total of 508 cases analyzed, 211 were aneuploid. All cases were correctly identified and no false results occurred (in comparison with karyotypic analysis), although maternal cell contamination was relatively common. CONCLUSION: Clinical screening for aneuploidy of chromosomes 13, 18, 21, X, and Y from interphase AF cells is possible with use of these probes and FISH. Cases of maternal cell contamination and mosaicism necessitate cautious interpretation. The FISH procedure is recommended for screening of common aneuploidies, followed by a complete chromosome analysis to detect anomalies.

Amniotic Fluid↗
PubMed2003

Comparison of interphase FISH and metaphase cytogenetics to study myelodysplastic syndrome: an Eastern Cooperative Oncology Group (ECOG) study.

Cytogenetic analysis can be important in determining the prognosis and diagnosis of a number of hematological disorders, including myelodysplastic syndromes (MDS). Here, we compared metaphase chromosomal analyses on bone marrow aspirates from MDS patients with interphase fluorescence in situ hybridization (FISH) using probes specific for chromosomes nos. 5, 7, 8, 11, 13 and 20. Forty-three patients enrolled in ECOG protocol E1996 for low risk MDS and five patients enrolled in ECOG protocol E3996 for high risk MDS were studied by both metaphase chromosomal analysis and interphase FISH. Excluding those with a clonal loss of the Y chromosome, an abnormal clone was detected by cytogenetic analysis in 18 of 48 samples (37.5%). In comparison, our FISH panel detected an abnormal clone in 17 of 48 samples (35.4%). Twenty-nine of 30 samples with apparently normal karyotypes, including those with a missing Y chromosome, were also normal by our FISH panel. One patient had an occult deletion of chromosome 11 that was detected by FISH. These results indicate that around 60% of patients with MDS do not have abnormalities that are detectable by either chromosomal or FISH studies. In addition, it appears that interphase FISH studies are nearly as sensitive as cytogenetic analyses and can be a useful tool in studying bone marrow aspirates where cytogenetic analysis is not possible.

Antineoplastic Agents↗
PubMed2001

Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma.

To detect immunoglobulin heavy chain (IGH) gene translocations with specific oncogene loci, we established an interphase cytogenetic approach using double-color fluorescence in situ hybridization (DC-FISH), which we used to analyze 173 patients with B-cell lymphoma. DC-FISH using the IGH gene (14q32.3) in combination with c-MYC (8q24.1), BCL1 (11q13.3), BCL2 (18q21.3), BCL6 (3q27), and PAX-5 (9p13) gene probes detected IGH translocations in 70 (40.5%) of 173 patients. The partner genes involved in IGH translocations were identified in 56 (80%) of 70 patients, and fusion of the IGH gene with specific oncogenes was detected in 53 of 56 patients, particularly in interphase nuclei of 28 patients for whom cytogenetic analysis was not informative. The most common partner gene was BCL2 (19 patients; 27% of IGH translocation-positive patients), followed by BCL6 (16; 23%), BCL1 (11; 16%), c-MYC (7; 10%), and PAX-5 (2; 3%). These oncogenes were closely associated with subtypes of B-cell lymphoma. The other partners were 19q13 (BCL3), 6p25 (MUM1/IRF4), 1q36, and chromosome 8 identified in one patient each. Six of the nine patients with add(14)(q32) showed a BCL6/IGH translocation. Double translocations of the IGH gene were found in three patients; c-MYC+BCL1, c-MYC+BCL2, and c-MYC+BCL6 in each one. Interphase FISH using specific IGH-translocation probes is valuable for defining clinically meaningful subgroups of B-cell lymphoma.

Adolescent↗
PubMed1999

AgNOR of human interphase cells in relation to acrocentric chromosomes.

Using simultaneous detection of fluorescence in situ hybridization (FISH) to acrocentric chromosome centromeres and argyrophilic nucleolar organizer regions (AgNOR), we investigated the number of AgNOR and involvement pattern of acrocentric chromosomes in the nucleoli in various types of human interphase cells. The number of AgNOR of normal gastric mucosal epithelial cells was 2.27 +/- 1.18 and was higher than that of lymphocytes (1.08 +/- 0.28) and lower than that of gastric cancer (7.76 +/- 3.21). The number of acrocentric chromosome centromere signals of normal gastric mucosal epithelial cells was higher than that of normal leukocytes (P < 0.000), and lower than that of gastric cancer (P < 0.000). The acrocentric chromosome centromere signals in the lymphocytes and neutrophils were only half of that expected for diploid cells, perhaps related to acrocentric chromosome association. The proportion of acrocentric chromosomes attached to AgNOR in gastric cancer (0.88 +/- 0.22) was significantly higher than that of normal gastric mucosal epithelial cells (0.72 +/- 0.35, P = 0.007). In conclusion, acrocentric chromosome association appears to be present in circulating leukocytes even in interphase. The number of AgNORs and proportion of acrocentric chromosomes involved in AgNORs in human interphase cells may vary according to cell types. This could play a significant role in rDNA transcription and determination of cell phenotype, including malignant change.

Cell Division↗
PubMed1999

Interphase fluorescence in situ hybridization overcomes pitfalls of G-banding analysis with special reference to underestimation of chromosomal aberration rates.

Fluorescence in situ hybridization (FISH) is suitable for detecting different types of chromosome aberrations on interphase nuclei even in specimens with no or few chromosome metaphases. However, it is not known why FISH is superior to conventional G-banding analysis. The sensitivity of interphase FISH was compared to that of G-banding analysis in 288 leukemia/lymphoma patients for 10 different types of chromosome aberrations: t(9;22) (M- and m-BCR), t(8;21), 11q23 abnormalities, t(15;17), del(5)/-5, del(13)/-13, +8, -7, and +12. The results revealed that t(15;17) positive cells could not proliferate well in culture, leading to underestimation of abnormality by G-banding. Monosomy 7 in acute myelocytic leukemia (AML) and myelodysplastic syndrome (MDS) as well as trisomy 12 and deletion chromosome 13 in chronic lymphocytic leukemias (CLL) were also severely underestimated by G-banding. On the other hand, no discrepancies were observed in t(8;21), t(9;22), translations involving 11q23, or in trisomy 8. These findings indicate the superiority of interphase FISH over conventional cytogenetics for detecting chromosome abnormalities in small clones, especially for monosomy 7 or (15;17) translocations.

Chromosome Aberrations↗
PubMed2001

Modelling chromosomal aberration induction by ionising radiation: the influence of interphase chromosome architecture.

Several advances have been achieved in the knowledge of nuclear architecture and functions during the last decade, thus allowing the identification of interphase chromosome territories and sub-chromosomal domains (e.g. arm and band domains). This is an important step in the study of radiation-induced chromosome aberrations; indeed, the coupling between track-structure simulations and reliable descriptions of the geometrical properties of the target is one of the main tasks in modelling aberration induction by radiation, since it allows one to clarify the role of the initial positioning of two DNA lesions in determining their interaction probability. In the present paper, the main recent findings on nuclear and chromosomal architecture are summarised. A few examples of models based on different descriptions of interphase chromosome organisation (random-walk models, domain models and static models) are presented, focussing on how the approach adopted in modelling the target nuclei and chromosomes can influence the simulation of chromosomal aberration yields. Each model is discussed by taking into account available experimental data on chromosome aberration induction and/or interphase chromatin organisation. Preliminary results from a mechanistic model based on a coupling between radiation track-structure features and explicitly-modelled, non-overlapping chromosome territories are presented.

Animals↗
PubMed2002

Localization of the gene-richest and the gene-poorest isochores in the interphase nuclei of mammals and birds.

At a resolution of 850 bands, human chromosomes comprise two subsets of bands, the GC-richest H3(+) and the GC-poorest L1(+) bands, accounting for about 17 and 26%, respectively, of all bands. The former are a subset of the R bands and the latter are a subset of the G bands. These bands showed the highest and the lowest gene densities, respectively, as well as a number of other distinct features. Here we report that human and chicken interphase nuclei are characterized by the following features. (1) The gene-richest/GC-richest chromosomal regions are predominantly distributed in internal locations, whereas the gene-poorest/GC-poorest DNA regions are close to the nuclear envelope. (2) The interphase chromosomes seem to be characterized by a polar arrangement, because the gene-richest/GC-richest bands and the gene-poorest/GC-poorest bands are predominantly located in the distal and proximal regions, respectively, of chromosomes, and because interphase chromosomes are extremely long. While this polar arrangement is evident in the larger chromosomes, it is not displayed by the chicken microchromosomes and by some small human chromosomes, namely by chromosomes that are almost only composed by GC-rich or by GC-poor DNA. (3) The gene-richest chromosomal regions display a much more spread-out conformation compared to the gene-poorest regions in human nuclei. This finding has interesting implications for the formation of GC-rich isochores of warm-blooded vertebrates.

Animals↗
PubMed1998

The DNA content of chromosome division figures and interphase nuclei classifies ulcerative colitis.

Long-standing ulcerative colitis is considered to be a precancerous condition. Therefore, a practical and reliable method is required for monitoring the progress of the disease. Liberation of the S-phase from karyokinesis occurs in DNA amplification and endoreplication, producing nuclei with more than 4 c DNA. The amount of Feulgen DNA was quantified with an image microphotometer in 8 microns sections for interphase nuclei and in 15 microns sections for chromosome division figures (CDFs). Development of ulcerative colitis was investigated in low grade dysplasia (n = 93 cases; score 3-7) and high grade dysplasia (n = 22; score 8-10). Bacterial colitis (n = 34) and invasive adenocarcinoma (n = 26) provided a basis for data interpretation in dysplasia. Lymphocyte nuclei served as an internal DNA standard. CDFs represent a novel type of aberrant 'mitoses'; they are different from and much more frequent than figures with multipolar spindles. Endoreplication began with low grade dysplasia in interphase nuclei as well as with CDFs; it was fully established in high grade dysplasia and carcinoma. Endoreplicated interphase nuclei and CDFs represent an early morphological mosaic of genomic instability. Both characteristics support a reproducible two-level classification of low and high grade dysplasia in ulcerative colitis.

Cell Nucleus↗
PubMed2000

Direct evidence for interphase chromosome movement during the mid-blastula transition in Drosophila.

In Drosophila, several genetic phenomena are most easily explained by a model in which homologous chromosomes pair, at least transiently, and use regulatory information present on only one homolog to pattern expression from both homologs [1] [2] [3]. To accomplish pairing of sites on different chromosomes, there must be a mechanism by which communication between homologs is facilitated. However, except in the case of meiotic prophase, directed, rapid chromosomal movement has not yet been observed. Some studies suggest that chromosomes are relatively immobile during interphase [4] [5], while others suggest that chromatin can reposition during interphase [6] [7] [8] and may be free to undergo substantial Brownian motion [9]. Using high-resolution, three-dimensional imaging techniques, we determined directly the structure and nuclear location of eleven different loci, both active and inactive, in embryos at cycle 14, the mid-blastula transition. We show that during a single interphase, portions of chromosomes moved in a cell-cycle-specific, directed fashion, independently and over long distances. All eleven regions showed movement, although the genes closer to the centromere moved faster (0.7 microm/minute) and over long distances (5-10 microm), whereas those nearer the telomere expanded in the same place and became oriented along the nuclear axis. Gene motion was independent of replication, transcription and changes in nuclear shape. Because individual genes on the same chromosome move independently, the movement is unlikely to be mediated by centromeres, Brownian motion or random drift and must be caused by an active mechanism.

Animals↗
PubMed2000

Dynamics of interphase microtubules in Schizosaccharomyces pombe.

BACKGROUND: Microtubules in interphase Schizosaccharomyces pombe are essential for maintaining the linear growth habit of these cells. The dynamics of assembly and disassembly of these microtubules are so far uncharacterised. RESULTS: Live cell confocal imaging of alpha1 tubulin tagged with enhanced green fluorescent protein revealed longitudinally oriented, dynamically unstable interphase microtubule assemblies (IMAs). The IMAs were uniformly bright along their length apart from a zone of approximately doubly intense fluorescence commonly present close to their centres. The ends of each IMA switched from growth ( approximately 3.0 microm/min) to shrinkage ( approximately 4.5 microm/min) at 1.0 events per minute and from shrinkage to growth at 1.9 events per minute, and the two ends were equivalently dynamic, suggesting equivalent structure. We accordingly propose a symmetrical model for microtubule packing within the IMAs, in which microtubules are plus ends out and overlap close to the equator of the cell. IMAs may contain multiple copies of this motif; if so, then within each IMA end, the microtubule ends must synchronise catastrophe and rescue. When both ends of an IMA lodge in the hemispherical cell ends, the IMAs start to bend under compression and their overall growth rate is inhibited about twofold. Similar microtubule dynamics were observed in cells ranging in size from half to twice normal length. Patterned photobleaching indicated no detectable treadmilling or microtubule sliding during interphase. CONCLUSIONS: The consequence of the mechanisms described is continuous recruitment of microtubule ends to the ends of growing cells, supporting microtubule-based transport into the cell ends and qualitatively accounting for the essential role for microtubules in directing linear cell growth in S. pombe.

Green Fluorescent Proteins↗
PubMed2001

Multiple regimes of constrained chromosome motion are regulated in the interphase Drosophila nucleus.

BACKGROUND: Increasing evidence indicates specific changes in the three-dimensional organization of chromosomes in the cell nucleus during the cell cycle and development. These changes may be linked to changes in both the coordinated regulation of gene transcription and the timing of chromosome replication. While there is cytological evidence for short-range diffusive motion of chromosomes during interphase, the mechanisms for large-scale chromosome remodeling inside the nucleus remain unknown. RESULTS: Chromosome motion was tracked in Drosophila spermatocyte nuclei by 3D fluorescence microscopy. The Lac repressor/lac operator system was used to label specific chromosomal sites in live tissues, allowing extended observation of chromatin motion in different cell cycle stages. Our results reveal a highly dynamic chromosome organization governed by two types of motion: a fast, short-range component over a 1-2 s time scale and a slower component related to long-range chromosome motion within the nucleus. The motion patterns are consistent with a random walk. In early G2, short-range motion occurs within a small, approximately 0.5 microm radius domain, while long-range motion is confined to a much larger, chromosome-sized domain. Progression through G2 as cells approach meiotic prophase is accompanied by a complete arrest of long-range chromosome motion. CONCLUSIONS: Our analysis provides direct evidence for cell cycle-regulated changes in interphase chromatin motion. These changes are consistent with changes in local and long-range constraints on chromosome motility. We propose that dynamic interactions between chromosomes and internal nuclear structures modulate the range and rate of interphase chromatin diffusion and thereby regulate large-scale nuclear chromosome organization.

Animals↗
PubMed1997

Interphase chromosomes undergo constrained diffusional motion in living cells.

BACKGROUND: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus. RESULTS: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture. CONCLUSIONS: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.

Animals↗
PubMed2004

[Topographical organisation of the chromatin in human interphase nuclei: architecture meets function].

There are an estimated number of 30,000 genes in the human genome, accounting for as few as 5% of the whole DNA content. Determining the exact role of the vast majority of untranscribed DNA is a major goal for upcoming years. Among various evolutionary constrains which could explain the presence of such a quantity of so-called "junk DNA", one hypothesis is the necessary controlled topographical arrangement of the genome during interphase, leading to a non-random, reproducible position of chromosomal regions inside the nucleus. This hypothesis relies on recent progresses in imaging technologies such as fluorescence confocal microscopy, allowing for the first time the identification of each chromosome-specific chromatin during interphase. This review focuses on the past years advances leading to the actual model of chromosome territories in the interphase nucleus.

Cell Nucleus↗
PubMed2003

Micronuclei, nuclear lesions and interphase silver-stained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill effluent.

In this study, cyto-genotoxic effects of a textile mill effluent on fish Oreochromis niloticus were investigated using the micronucleus (MN) test and methods to analyze interphase silver-stained nucleolar organizer regions (AgNORs). Fishes were exposed to three different concentrations of textile mill effluent (5, 10 and 20%(v/v)) for 3, 6 and 9 days. Cyclophosphamide (2mg/l) was used as a positive control. Micronucleus frequencies were examined in peripheral blood erythrocytes and gill cells. Nuclear abnormalities (NA) other than micronuclei such as binuclei, lobed nuclei, blebbed nuclei and notched nuclei were also evaluated in peripheral erythrocytes. Interphase AgNOR parameters were examined in epithelial cells obtained from the edge of caudal fins after 90 and 180min of exposure. As a result, dose-dependent increases in the frequencies of micronuclei and other NA in erythrocytes were observed. MN frequencies in gill cells also significantly increased, while the interphase AgNOR parameters in fin cells decreased, as a result of textile effluent and cylophosphamide treatments.

Animals↗
PubMed1985

Decrease in ADP-ribosylation of HeLa non-histone proteins from interphase to metaphase.

Variations for non-histones in the ADP-ribosylating activities of interphase and metaphase cells were investigated. 32P-Labeled nicotinamide adenine dinucleotide ([32P]NAD), the specific precursor for the modification, was used to radioactively label proteins. Permeabilized interphase and mitotic cells, as well as isolated nuclei and chromosomes, were incubated with the label. One-dimensional and two-dimensional gels of the proteins of total nuclei and chromatin labeled with [32P]NAD showed more than 100 modified species. Changing the labeling conditions resulted in generally similar patterns of modified proteins, though the overall levels of incorporation and the distributions of label among species were significantly affected. A less complex pattern was found for nuclear scaffolds. The major ADP-ribosylated proteins included the lamins and poly(ADP-ribose) polymerase. Inhibitors of ADP-ribosylation were effective in preventing the incorporation of label by most non-histones. Snake venom phosphodiesterase readily removed protein-bound 32P radioactivity. A fundamentally different distribution of label from that of interphase nuclei and chromatin was found for metaphase chromosome non-histones. Instead of 100 or more species, the only major acceptor of label was poly(ADP-ribose) polymerase. This profound change during mitosis may indicate a structural role for ADP-ribosylation of non-histone proteins.

Adenosine Diphosphate Ribose↗
PubMed2002

Cnd2 has dual roles in mitotic condensation and interphase.

Chromosome condensation requires condensin, which comprises five subunits. Two of these subunits--both being structural maintenance of chromosome (SMC) proteins-are coiled-coils with globular terminal domains that interact with ATP and DNA. The remaining three, non-SMC subunits also have essential, albeit undefined, roles in condensation. Here we report that Cnd2 (ref. 6), a non-SMC subunit of fission yeast similar to Drosophila Barren and the budding yeast protein Brn1 (refs 8, 9), is required for both interphase and mitotic condensation. In cnd2-1 mutants, ultraviolet-induced DNA damage is not repaired, and cells arrested by hydroxyurea do not recover. A definitive defect of interphase is abolishment of Cds1 (a checkpoint kinase) activation in the presence of hydroxyurea in both cnd2-1 mutant cells and in cells where other condensin subunits have been genetically disrupted. In the absence of hydroxyurea, a G2 checkpoint delay occurred in cnd2-1 mutants in a manner dependent on Cds1 and ATM-like Rad3, but not Chk1 (refs 10-13), before the mitotic condensation defect. Furthermore, cnd2-1 was synthetic-lethal with mutations of excision repair, RecQ helicase and DNA replication enzymes. These interphase and mitotic defects provide insight into the mechanistic role of non-SMC subunits that interact with the globular SMC domains in the heteropentameric holocomplex.

Cell Cycle Proteins↗
PubMed2005

Sequential application of interphase-FISH and CGH to single cells.

A comprehensive genomic analysis of single cells is needed for numerous scenarios in tumor genetics, clinical diagnostics and forensic application. PCR protocols were developed which allow an unbiased amplification of the whole genome of a single cell for subsequent analyses by comparative genomic hybridization (CGH). However, verification of single-cell CGH results has been impossible as the procedure naturally involves the destruction of the respective cell. Here we show that the genome of individual cells can be analyzed by two different single cell techniques applied sequentially to the same cell. In a first step, interphase fluorescence in situ hybridization (FISH) is applied. After evaluation of the interphase-FISH signals, cells of interest can be selected for a further analysis. Single cells are collected by laser microdissection, the DNA is amplified by linker-adaptor PCR and subjected to CGH-analysis. This strategy offers new opportunities for a sophisticated selection of cells based on interphase-FISH signals. Furthermore, the sequential application of two different single-cell approaches to the same single-cell represents the only option to control and verify the single-cell CGH results. We demonstrate the feasibility of this approach with a series of experiments including cells from pre- and postnatal diagnostics, for example, cells with trisomies 13, 18, or 21, respectively, leukemia and tumor cells and tissue sections.

In Situ Hybridization, Fluorescence↗