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PubMed1998

Isolation and characterization of fission yeast sns mutants defective at the mitosis-to-interphase transition.

pim1-d1ts was previously identified in a visual screen for fission yeast mutants unable to complete the mitosis-to-interphase transition. pim1+ encodes the guanine nucleotide exchange factor (GEF) for the spi1 GTPase. Perturbations of this GTPase system by either mutation or overproduction of its regulatory proteins cause cells to arrest with postmitotic condensed chromosomes, an unreplicated genome, and a wide medial septum. The septation phenotype of pim1-d1ts was used as the basis for a more extensive screen for this novel class of sns (septated, not in S-phase) mutants. Seventeen mutants representing 14 complementation groups were isolated. Three strains, sns-A3, sns-A5, and sns-A6, representing two different alleles, are mutated in the pim1+ gene. Of the 13 non-pim1ts sns complementation groups, 11 showed genetic interactions with the spi1 GTPase system. The genes mutated in 10 sns strains were synthetically lethal with pim1-d1, and six sns strains were hypersensitive to overexpression of one or more of the known components of the spil GTPase system. Epistasis analysis places the action of the genes mutated in nine of these strains downstream of pim1+ and the action of one gene upstream of pim1+. Three strains, sns-A2, sns-B1, and sns-B9, showed genetic interaction with the spil GTPase system in every test performed. sns-B1 and sns-B9 are likely to identify downstream targets, whereas sns-A2 is likely to identify upstream regulators of the spi1 GTPase system that are required for the mitosis-to-interphase transition.

Alleles↗
PubMed2001

Fission yeast Mog1p homologue, which interacts with the small GTPase Ran, is required for mitosis-to-interphase transition and poly(A)(+) RNA metabolism.

We have cloned and characterized the Schizosaccharomyces pombe gene mog1(+), which encodes a protein with homology to the Saccharomyces cerevisiae Mog1p participating in the Ran-GTPase system. The S. pombe Mog1p is predominantly localized in the nucleus. In contrast to the S. cerevisiae MOG1 gene, the S. pombe mog1(+) gene is essential for cell viability. mog1(+) is required for the mitosis-to-interphase transition, as the mog1-1 mutant arrests at restrictive temperatures as septated, binucleated cells with highly condensed chromosomes and an aberrant nuclear envelope. FACS analysis showed that these cells do not undergo a subsequent round of DNA replication. Surprisingly, also unlike the Delta mog1 mutation in S. cerevisiae, the mog1-1 mutation causes nucleolar accumulation of poly(A)(+) RNA at the restrictive temperature in S. pombe, but the signals do not overlap with the fibrillarin-rich region of the nucleolus. Thus, we found that mog1(+) is required for the mitosis-to-interphase transition and a class of RNA metabolism. In our attempt to identify suppressors of mog1-1, we isolated the spi1(+) gene, which encodes the fission yeast homologue of Ran. We found that overexpression of Spi1p rescues the S. pombe Delta mog1 cells from death. On the basis of these results, we conclude that mog1(+) is involved in the Ran-GTPase system.

Active Transport, Cell Nucleus↗
PubMed1997

Carrier-specific breakpoint-spanning DNA probes: an approach to preimplantation genetic diagnosis in interphase cells.

Carriers of chromosomal inversions or other balanced rearrangements represent a significant fraction of patients in in-vitro fertilization (IVF) programmes due to recurrent reproductive problems. In most cases, chromosomal imbalance in fertilized oocytes is incompatible with embryo survival leading to increased rates of spontaneous abortions. Assuming that a fraction of the germ cells is karyotypically normal, these patients would greatly benefit from efficient procedures for generation and use of breakpoint-specific DNA hybridization probes in preconception and preimplantation genetic diagnosis (PGD). We describe the generation of such patient-specific probes to discriminate between normal and aberrant chromosomes in interphase cells. First, a large insert DNA library was screened for probes that bind adjacent to the chromosomal breakpoints or span them. Then, probe and hybridization parameters were optimized using white blood cells from the carrier to increase in hybridization signal intensity and contrast. Finally, the probes were tested on target cells (typically polar bodies or blastomeres) and a decision about the colour labelling scheme was made, before the probes can be used for preconception or preimplantation genetic analysis. Thus, it was demonstrated that cells with known structural abnormalities could be detected, based on hybridization of breakpoint spanning yeast artificial chromosome (YAC) DNA probes in interphase cells.

Blastomeres↗
PubMed1994

Optimal preparation of preimplantation embryo interphase nuclei for analysis by fluorescence in-situ hybridization.

A new method was developed to prepare and isolate interphase nuclei from murine preimplantation embryos for analysis by fluorescence in-situ hybridization. Embryos were washed with phosphate-buffered saline and disaggregated in a small drop of bidistilled water containing HCl and Tween 20. During the disaggregation procedure embryos were watched continuously with an inverted microscope. Embryonic nuclei were digested with pepsin to make them accessible for hybridization to the probes and to remove remnants of cytoplasm. Nuclear and probe DNA were denatured simultaneously and hybridization was done overnight, followed by immunocytochemical detection of the probes. Using this method we were able to perform successful in-situ hybridization on all preimplantation stages of the mouse embryo (pronuclei, 2-cell, 4-cell, morula and blastocyst). Probes for the X and Y chromosomes were applied for sex determination. From the results described in this paper we conclude that the preparation and isolation of interphase nuclei from murine embryos with acid and Tween 20 offers high reproducibility, good morphology of the cells and a high hybridization efficiency.

Animals↗
PubMed2002

Biophysical study of the globular organisation of interphase chromosomes.

The globular model of interphase chromosomes is studied using methods involving the statistical physics of polymers. An interphase chromatid is represented as a flexible chain of structural sub-units, or superdomains (SDs). Each SD is simulated as a number of chromatin fibre loops fixed at a nuclear matrix core. A chain of SDs is further folded in the nucleus in a compact conformation owing to volumetric interactions between SDs. The algorithm used is extended to incorporate the chain anchorage at different points. Excluded volume effects are taken into account in Monte Carlo simulation at both the SD and whole chromosome level. A variety of structures is observed in computer experiments. The simulation results correlate with the available experimental data.

Biophysics↗
PubMed1997

Detection of chromosomal changes by interphase cytogenetics in biopsies of recurrent astrocytomas and oligodendrogliomas.

Recently, lineage-specific genetic pathways of tumor progression have been suggested in both oligodendrogliomas and astrocytomas. Aberrations consistently reported in gliomas include chromosomes 1, 7, 10, 17 and 19. Identification of specific genetic damage may have important clinical consequences, because oligodendrogliomas, unlike astrocytomas, are responsive to chemotherapy. Genetic alterations specific for tumor type and tumor progression were investigated in 5 pairs of recurrent astrocytomas and 8 pairs of recurrent oligodendrogliomas by means of interphase in situ hybridization (ISH) to paraffin-embedded, formalin-fixed tissue sections. A set of DNA probes specific for the centromeric regions of chromosomes 1, 7, 10, 17, X and Y was applied. Since LOH studies on oligodendrogliomas have revealed losses in the region of 1p32-1p36, a DNA probe specific for the 1p36.3 locus was included. Hybridization with the 1p36.3 probe revealed loss of 1p in 5 of the 8 oligodendroglioma recurrences, the aberration being present in the primary tumors in 2 cases. In none of the astrocytomas was loss of 1p observed. Numerical aberrations were found in one astrocytoma pair (+7) and in the second biopsy of an oligodendroglioma (+7, -10). Aneuploidy was found by in situ hybridization in 8 of the 13 tumor pairs. Detection of aberrations in the 1p36.3 locus by interphase in situ hybridization to paraffin-embedded, formalin-fixed tumors may become a very useful tool in delineation of oligodendroglial from astrocytic genotypes, directing tumor specific therapy. The technique may be of crucial importance in tumor cases in which histologic criteria of lineage are not obvious.

Adult↗
PubMed2006

The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material.

Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environment. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environment that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of chromosomes at metaphase may underestimate their genotoxic potential. Premature Chromosome Condensation (PCC) induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G1 and G2 phases of the cell cycle. The PCC has been successfully used in problems involving cell cycle analysis, diagnosis and prognosis of human leukaemia, assessment of interphase chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in answering questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics and experimental therapeutics.

Animals↗
PubMed2005

Uniparental chromosome elimination at mitosis and interphase in wheat and pearl millet crosses involves micronucleus formation, progressive heterochromatinization, and DNA fragmentation.

Complete uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanisms underlying selective elimination of the paternal chromosomes during the development of wheat (Triticum aestivum) x pearl millet (Pennisetum glaucum) hybrid embryos. All pearl millet chromosomes were eliminated in a random sequence between 6 and 23 d after pollination. Parental genomes were spatially separated within the hybrid nucleus, and pearl millet chromatin destined for elimination occupied peripheral interphase positions. Structural reorganization of the paternal chromosomes occurred, and mitotic behavior differed between the parental chromosomes. We provide evidence for a novel chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei is different, and heterochromatinization and DNA fragmentation of micronucleated pearl millet chromatin is the final step during haploidization.

Chromosomes, Plant↗
PubMed2004

Interphase chromosomes and the Rabl configuration: does genome size matter?

Summary It is now well established that the cereals share a common gene order or gene synteny. However, the cereal species encompass an enormous range of genome size, with wheat being one of the largest and rice one of the smallest. Here we describe the current state of knowledge of interphase chromosome structure within the cereal species. In wheat and its close relatives, the interphase chromosomes adopt a highly regular Rabl configuration, with the two chromosome arms lying next to each other and the centromeres and telomeres located at opposite poles of the nuclei. By contrast, the chromosomes in most rice nuclei clearly do not show a Rabl configuration. Surprisingly, the chromosomes in the endoreduplicated xylem vessel cells of rice do adopt a Rabl configuration. To explain this observation, we propose that endoreduplication may occur immediately after chromosome segregation in these cells, and that the new chromatin interactions, particularly at the centromeres, in the endoreduplicated chromosomes may stabilize the anaphase chromosome configuration.

Chromosome Pairing↗
PubMed1992

Histological features and interphase nucleolar organizer regions in hyperplastic, dysplastic and neoplastic epithelium of intrahepatic bile ducts in hepatolithiasis.

Neoplastic transformation occurs in the intrahepatic biliary tree in hepatolithiasis. The present study aimed to clarify the neoplastic processes by correlating the histological features of the bile duct lesions with counts of interphase argyrophilic nucleolar organizer regions (AgNORs), which reflect cell proliferative activity. We studied 55 cases of hepatolithiasis and 25 normal autopsy livers. The biliary epithelial lesions in hepatolithiasis were divisible into hyperplasia, dysplasia and neoplasia. These lesions were found in bile ducts containing calculi. All cases of hepatolithiasis showed a varied degree of hyperplasia. Additionally, eight cases showed dysplasia, five non-invasive intraductal adenocarcinoma and 10 invasive adenocarcinoma. Cases of non-invasive and invasive carcinoma frequently harboured areas of dysplasia, and areas of dysplasia and non-invasive carcinoma, respectively. The mean and standard deviation of the number of interphase AgNORs in the normal and abnormal biliary epithelium showed a step-wise increase in the following order: normal (1.32 +/- 0.36), hyperplasia (1.52 +/- 0.37), dysplasia (2.28 +/- 0.56), non-invasive carcinoma (3.23 +/- 1.00), and invasive carcinoma (3.72 +/- 0.77). These histological and cell kinetic observations suggest that, in hepatolithiasis, carcinogenesis in bile duct epithelial cells progresses in a multi-step manner, through hyperplasia, dysplasia, non-invasive adenocarcinoma and invasive adenocarcinoma.

Adenoma, Bile Duct↗
PubMed1995

Complete hydatidiform mole in twin pregnancy: differentiation from partial mole with interphase cytogenetic and DNA cytometric analyses on paraffin embedded tissues.

Six cases of hydatidiform mole associated with normal chorionic villi and a normal embryo/fetus (in five cases) were investigated with interphase cytogenetic and DNA cytometric analyses for diagnostic purposes. DNA probes specific for the pericentromeric regions of chromosomes 1 and X and for the long arm of chromosome Y were used. In four cases a dizygotic twin pregnancy could be proven. In these cases, the histologically normal chorionic villi showed an XY DNA-diploid pattern, consistent with a normal male conceptus, and the molar chorionic villi a XX pattern. In the other two cases an identical sex chromosomal pattern was found in the normal and in the molar villi (XX/XX and XY/XY respectively). In all six cases the molar placental tissues showed prominent trophoblastic hyperplasia with DNA-polyploidy, consistent with a complete hydatidiform mole. In two cases persistent gestational trophoblastic disease developed. It is emphasized that twin pregnancies composed of a normal conceptus and a complete mole have a relatively high risk for the development of persistent trophoblastic disease and therefore, should be carefully differentiated from triploid partial moles with a relatively low risk of persistent gestational trophoblastic disease. These case reports indicate that additional interphase cytogenetic and DNA cytometric analyses are useful in this differential diagnosis.

Adult↗
PubMed2006

Blastoid and common variants of mantle cell lymphoma exhibit distinct immunophenotypic and interphase FISH features.

AIMS: The recognition of blastoid variant (BV) of mantle cell lymphoma (MCL) is based on morphological criteria. Our aim was to analyse 18 MCL cases including four BV-MCL for their clinicopathological features, proliferation index, cyclin D1 and CDK4 expression and interphase fluorescence in-situ hybridization (FISH) pattern. METHODS AND RESULTS: BV-MCL versus common MCL was characterized by a shorter overall duration of response after first-line therapy (11 months versus 28 months) and shorter overall survival (20 months versus 42 months). Interphase FISH showed a t(11;14) fusion pattern in all MCL tested cases. However, the four blastoid cases were characterized by extra copies of CCND1 signals. Using additional probes of chromosomes 11, 18, 21, these signals were shown to be the result of hypotetraploidy and not of a specific amplification of the normal or the translocated CCND1 allele. Moreover, the BV-MCL cases were characterized by a combined high percentage of cells expressing cyclin D1 and/or CDK4 with a proliferation (MIB-1-Ki67) index above 50%. Such features allowed the recognition of areas of large cell transformation in the case of secondary BV-MCL. CONCLUSIONS: Since distinction between BV and common MCL is of clinical relevance, our data underline the need to add phenotypic and cytogenetic criteria to cytomorphology for a better recognition of BV-MCL.

Adult↗
PubMed1986

The presence of intracytoplasmic confronting cisternae of the endoplasmic reticulum in osteosarcoma cells in interphase.

Confronting cisternae of the endoplasmic reticulum recognized in tumor cells of 7 cases of osteosarcoma were presented. They were found in the mitotic cells as well as in the cytoplasms of interphase cells. The more the mitotic cells were observed in 1 micron-thick sections, the more frequently those membranous structures were encountered in the corresponding ultrathin sections. In the interphase cells, such structures were located around Golgi apparatus or close to the nucleus. Occasionally, they were composed of a pair of closely apposed cisternae of the nuclear membrane and the rough endoplasmic reticulum. These results seem to indicate that the nuclear envelope which is disrupted and reformed during mitosis in rapidly proliferating cells takes part in the formation of the confronting cisternae of the endoplasmic reticulum.

Adolescent↗
PubMed1992

Interphase cytogenetics of the ICF syndrome.

Interphase behaviour of centromeric heterochromatin of chromosomes 1 and 16 has been investigated in lymphocytes and fibroblasts of patients with ICF syndrome and of normal subjects with non-isotopic in situ hybridization, using the satellite II-related probe pHuR 195. We found evidence for interphase somatic pairing in ICF lymphocytes with a frequency higher than that found in normal cells. Lymphocytes of ICF patients showed nuclear protrusions and micronuclei and these nuclear abnormalities consistently involved a hybridization signal. Somatic pairing was also present in fibroblasts, but with frequencies similar in normal and ICF subjects. The fibroblasts do not have the major chromosomal abnormalities found in lymphocytes. The degree of heterochromatin condensation in fibroblasts was lower than that in lymphocytes and we postulate that the more decondensed state of chromocentres in the fibroblasts could be the reason for the absence of the major chromosomal abnormalities.

Cell Nucleus↗
PubMed1985

Indications of centromere movement during interphase and differentiation.

Mouse and human DNA sequences from centromeric and ribosomal domains were labeled with biotinylated deoxynucleotides and hybridized in situ to paraformaldehyde-fixed tissue culture cells. Centromeres were widely dispersed in most of these interphase nuclei. At late G2 phases of the cell cycle, centromeres appeared to coalesce and then to align in an orderly pattern, with discrete positional assignments for individuals chromosomes in metaphase and anaphase. Ribosomal cistrons were also organized in an orderly and defined fashion during mitosis. As soon as the nuclear membrane forms in early G1, centromeres rapidly disperse throughout the nucleus. Centromere patterns during G1 and S were indistinguishable in cultured cells, as determined by double-labeling experiments. Antibodies that bind to centric chromosomal proteins revealed the same patterns in cultured cells as those obtained with DNA sequence-specific probes. Large differentiated neurons display reproducible collections of centromeres in interphase that are very different from those seen in cultured cells. Neurons in widely divergent mammalian species, despite large differences in centromeric DNA sequences, maintain similar nuclear positions for these chromosomal segments. Similarly, ribosomal cistrons are positioned in comparable nuclear locales in neurons of divergent species. It is suggested that such arrangements reflect, or are necessary for, the function of a given cell type. Studies of large cerebellar neurons at critical times in development indicated a relative "movement" of centromeric domains, away from the nuclear membrane and toward the central nucleolar region. It is possible that the orderly and temporal positioning of centromeric, as well as of other chromosomal regions, is based on protein-nucleic acid interactions. Implications for trisomy 21 and other disorders involving chromosomal rearrangements, such as transposition, are considered from this perspective.

Animals↗
PubMed1990

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line.

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.

Carbohydrate Metabolism↗
PubMed2002

Visualizing chromatin dynamics in interphase nuclei.

Real-time fluorescence microscopy has emerged as a powerful tool for examining chromatin dynamics. The initial lesson is that much of the genome, particularly in yeast, is highly dynamic. Its movement within the interphase nucleus is correlated with metabolic activity. Nonetheless, the nucleus is an organelle with conserved rules of organization. Determining the distribution and regulation of mobile domains in interphase chromosomes, and characterizing sites of anchorage, will undoubtedly shed new light on the function of nuclear order.

Animals↗
PubMed2002

Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts.

The small guanosine triphosphatase Ran is loaded with guanosine triphosphate (GTP) by the chromatin-bound guanine nucleotide exchange factor RCC1 and releases import cargoes in the nucleus during interphase. In mitosis, Ran-GTP promotes spindle assembly around chromosomes by locally discharging cargoes that regulate microtubule dynamics and organization. We used fluorescence resonance energy transfer-based biosensors to visualize gradients of Ran-GTP and liberated cargoes around chromosomes in mitotic Xenopus egg extracts. Both gradients were required to assemble and maintain spindle structure. During interphase, Ran-GTP was highly enriched in the nucleoplasm, and a steep concentration difference between nuclear and cytoplasmic Ran-GTP was established, providing evidence for a Ran-GTP gradient surrounding chromosomes throughout the cell cycle.

Active Transport, Cell Nucleus↗