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PubMed2005

The kinesin Klp2 mediates polarization of interphase microtubules in fission yeast.

Fission yeast (Schizosaccharomyces pombe) cells grow longitudinally in a manner dependent on a polarized distribution of their interphase microtubules. We found that this distribution required sliding of microtubules toward the cell center along preexisting microtubules. This sliding was mediated by the minus end-directed kinesin motor Klp2, which helped microtubules to become properly organized with plus ends predominantly oriented toward the cell ends and minus ends toward the cell center. Thus, interphase microtubules in the fission yeast require motor activities for their proper organization.

Benzimidazoles↗
PubMed1990

A view of interphase chromosomes.

Metaphase chromosomes are dynamically modified in interphase. This review focuses on how these structures can be modified, and explores the functional mechanisms and significance of these changes. Current analyses of genes often focus on relatively short stretches of DNA and consider chromatin conformations that incorporate only a few kilobases of DNA. In interphase nuclei, however, orderly transcription and replication can involve highly folded chromosomal domains containing hundreds of kilobases of DNA. Specific "junk" DNA sequences within selected chromosome domains may participate in more complex levels of chromosome folding, and may index different genetic compartments for orderly transcription and replication. Three-dimensional chromosome positions within the nucleus may also contribute to phenotypic expression. Entire chromosomes are maintained as discrete, reasonably compact entities in the nucleus, and heterochromatic coiled domains of several thousand kilobases can acquire unique three-dimensional positions in differentiated cell types. Some aspects of neoplasia may relate to alterations in chromosome structure at several higher levels of organization.

Animals↗
PubMed1996

Requirement for cAMP-PKA pathway activation by M phase-promoting factor in the transition from mitosis to interphase.

Cell cycle progression in cycling Xenopus egg extracts is accompanied by fluctuations in the concentration of adenosine 3',5'-monophosphate (cAMP) and in the activity of the cAMP-dependent protein kinase (PKA). The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase. Blocking the activation of PKA at metaphase prevented the transition into interphase; the activity of M phase-promoting factor (MPF; the cyclin B-p34cdc2 complex) remained high, and mitotic cyclins were not degraded. The arrest in mitosis was reversed by the reactivation of PKA. The inhibition of protein synthesis prevented the accumulation of cyclin and the oscillations of MPF, PKA, and cAMP. Addition of recombinant nondegradable cyclin B activated p34cdc2 and PKA and induced the degradation of full-length cyclin B. Addition of cyclin A activated p34cdc2 but not PKA, nor did it induce the degradation of full-length cyclin B. These findings suggest that cyclin degradation and exit from mitosis require MPF-dependent activation of the cAMP-PKA pathway.

Animals↗
PubMed1999

Cytoplasmic localization of human cdc25C during interphase requires an intact 14-3-3 binding site.

cdc25C induces mitosis by activating the cdc2-cyclin B complex. The intracellular localization of cyclin B1 is regulated in a cell cycle-specific manner, and its entry into the nucleus may be required for the initiation of mitosis. To determine the cellular localization of cdc25C, monoclonal antibodies specific for cdc25C were developed and used to demonstrate that in human cells, cdc25C is retained in the cytoplasm during interphase. A deletion analysis identified a 58-amino-acid region (amino acids 201 to 258) in cdc25C that was required for the cytoplasmic localization of cdc25C. This region contained a specific binding site for 14-3-3 proteins, and mutations in cdc25C that disrupted 14-3-3 binding also disrupted the cytoplasmic localization of cdc25C during interphase. cdc25C proteins that do not contain a binding site for 14-3-3 proteins showed a pancellular localization and an increased ability to induce premature chromosome condensation. The cytoplasmic localization of cdc25C was not altered by gamma irradiation or treatment with the nuclear export inhibitor leptomycin B. These results suggest that 14-3-3 proteins may negatively regulate cdc25C function by sequestering cdc25C in the cytoplasm.

Antibodies, Monoclonal↗
PubMed2005

The pattern of chromosome folding in interphase is outlined by the linear gene density profile.

Spatial organization of chromatin in the interphase nucleus plays a role in gene expression and inheritance. Although it appears not to be random, the principles of this organization are largely unknown. In this work, we show an explicit relationship between the intranuclear localization of various chromosome segments and the pattern of gene distribution along the genome sequence. Using a 7-megabase-long region of the Drosophila melanogaster chromosome 2 as a model, we observed that the six gene-poor chromosome segments identified in the region interact with components of the nuclear matrix to form a compact stable cluster. The six gene-rich segments form a spatially segregated unstable cluster dependent on nonmatrix nuclear proteins. The resulting composite structure formed by clusters of gene-rich and gene-poor regions is reproducible between the nuclei. We suggest that certain aspects of chromosome folding in interphase are predetermined and can be inferred through in silico analysis of chromosome sequence, using gene density profile as a manifestation of "folding code."

Animals↗
PubMed1989

Interphase cytogenetics using biotin and digoxigenin labelled probes II: Simultaneous differential detection of human and papilloma virus nucleic acids in individual nuclei.

A method was developed for the simultaneous detection of viral and human DNA in contrasting colours in routine formalin fixed, paraffin wax embedded biopsy specimens. This was achieved by non-isotopic in situ hybridisation (NISH) with a biotinylated Y chromosome probe and digoxigenin labelled probe for human papilloma virus type 6 (HPV 6). The tissues studied were peripheral lymphocytes, tonsil, and penile warts. The hybridisation signals produced by biotinylated probes were visualised in red using streptavidin peroxidase and those produced by digoxigenin labelled probes as a blue/black colour using anti-digoxigenin alkaline phosphatase. In lymphocytes and tonsil 95-100% of cells had a detectable Y chromosome; in warts only 60-70% of infected keratinocytes near the skin surface had a demonstrable Y chromosome. This suggests that this chromosome is lost or occluded in cell maturation. In simultaneous double hybridisation with both probes, HPV and Y sequences were demonstrable within the same nucleus in penile warts. This technique permits the simultaneous differential detection of two nuclei acid sequences in interphase nuclei and will have application in analysis of putative dual HPV infections and in determining the intranuclear spatial relations between nucleic acids in interphase nuclei.

Biotin↗
PubMed1998

Interphase cytogenetic and AgNOR analyses of hydatidiform moles.

AIM: To determine the potential value of interphase cytogenetic and argyrophilic nucleolar organiser region (AgNOR) analyses in the diagnosis and classification of hydatidiform moles. METHODS: Serial tissue sections from 37 hydatidiform moles, histologically classified as 11 complete and 15 partial, and from 11 hydropic abortuses were examined by in situ hybridisation using digoxigenin labelled probes specific for chromosomes 1, X, and Y, and a one step silver staining method. The percentages of diploid and triploid nuclei, and the mean number of AgNORs for each tissue were determined. RESULTS: Interphase cytogenetics showed that eight of the 11 cases (73%) each of complete mole and hydropic abortus had diploid pattern and the three remaining cases (27%) of each group were triploid. Two of the triploid complete moles and one of the triploid hydropic abortuses were revised to partial moles and one remaining triploid complete mole was revised to hydropic abortus. Of the 15 partial moles, nine (60%) were triploid, and six (40%) were diploid. These diploid cases were revised to three complete moles and three hydropic abortuses. There was a significant difference (p < 0.0001) between the mean (SD) AgNOR count in partial mole (5.11 (0.91)) versus hydropic abortus (3.79 (0.90)) and complete mole (3.39 (0.97)). The total of 15 triploid cases showed a high mean AgNOR count of 5.24 (0.73). Also, after reclassification, eight of the nine partial moles (89%) had a mean AgNOR count of > or = 5. The results of analyses by the two methods were closely correlated. CONCLUSIONS: Interphasecytogeneticanalysis using chromosome specific probes and AgNOR count provides a valuable approach for ploidy analysis in histological sections of hydatidiform moles and helps to resolve difficult cases.

Analysis of Variance↗
PubMed2006

Interphase fluorescence in situ hybridization analysis of chromosome 12p abnormalities is useful for distinguishing epidermoid cysts of the testis from pure mature teratoma.

PURPOSE: The distinction of epidermoid cyst of the testis from teratoma is of critical importance because the former is benign and the latter is a malignant tumor that may have associated metastasis of either teratomatous or non-teratomatous germ cell tumor types. Chromosome 12p abnormalities are seen in the vast majority of testicular germ cell tumors of adults and are present in all histologic subtypes. In this study, we investigated the clinical utility of interphase fluorescence in situ hybridization (FISH) analysis of chromosome 12p abnormalities for distinguishing epidermoid cysts of the testis from pure mature teratoma. EXPERIMENTAL DESIGN: Sixteen testicular epidermoid cysts and 17 testicular teratomas were investigated for isochromosome 12p [i(12p)] and 12p overrepresentation using interphase FISH analysis. RESULTS: Neither i(12p) nor 12p overrepresentation were observed in 16 epidermoid cyst cases, whereas i(12p) was detected in 76% of teratomas and 12p overrepresentation was identified in 29% of teratomas. Overall, 88% of testicular teratomas had chromosome 12p abnormalities. CONCLUSIONS: FISH identification of i(12p) and/or 12p overrepresentation in routinely processed surgical specimens is a useful ancillary diagnostic tool in distinguishing testicular epidermoid cysts from teratoma.

Adolescent↗
PubMed2000

RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH.

Activation of the RET protooncogene through somatic rearrangements represents the most common genetic alteration in papillary thyroid carcinoma (PTC). Three main rearranged forms of RET have been described: RET/PTC1 and RET/PTC3, which arise from a paracentric inversion of the long arm of chromosome 10, and RET/PTC2, which originates from a 10;17 translocation. We have developed a dual-color FISH approach to detect RET/PTC rearrangements in interphase nuclei of thyroid lesions. By using a pool of three cosmids encompassing the RET chromosome region and a chromosome 10 centromeric probe, we could discriminate between the presence of an inversion (RET/PTC1 and RET/PTC3) or a translocation (RET/PTC2). We have investigated a series of thyroid tissue samples from Italian and French patients corresponding to a total of 69 PTCs and 22 benign lesions. Among PTCs, 13 (18.8%) showed a RET rearrangement, and 11 (15.9%) of these carried an inversion (RET/PTC1 or RET/PTC3) in more than 10% of the nuclei examined. Activated forms of RET were also observed in three adenomas. RT-PCR analysis on the same samples confirmed the presence and the type of rearrangement predicted using FISH analysis. An interesting difference in the frequency and type of RET rearrangements was detected between the Italian and the French patients. Furthermore, we identified a putative novel type of rearrangement in at least one PTC sample. Several PTCs carried a significant number of cells characterized by a trisomy or a tetrasomy of chromosome 10. Overall, the FISH approach in interphase nuclei represents a powerful tool for detecting, at the single cell level, RET/PTC rearrangements and other anomalies involving the RET chromosome region.

Adenoma↗
PubMed2006

Detection of B chromosomes in interphase hemolymph nuclei from living specimens of the grasshopper Eyprepocnemis plorans.

The two most important evolutionary properties of B chromosomes are their transmission rate (which suggests their selfishness when significantly higher than 0.5) and their net effects on carrier fitness (usually negative for parasitic Bs). The study of transmission rate unavoidably requires the analysis of many controlled crosses in order to accurately measure population average transmission rate. Therefore, getting a marker closely associated to B presence is of crucial importance to alleviate the load of performing many useless crosses between lacking B individuals. After investigating several cytogenetic techniques on several tissues that may be sampled without drastically damaging live specimens of the grasshopper Eyprepocnemis plorans, we report here the excellent results provided by the CMA3 fluorescence and C-banding techniques applied to hemocyte nuclei. These cells may be easily obtained from both males and females and provide information on B presence even during the interphase stage. The two cytogenetic techniques take advantage of the heterochromatic nature of the B chromosomes, so that Bs made predominantly of ribosomal DNA are revealed by CMA3 as bright bodies in the interphase hemocytes, and Bs mostly made of satellite DNA are visualized by C-banding as intensely stained bodies in these cells.

Animals↗
PubMed1978

The nature of the Ag-staining of nucleolus organizer regions. Electron- and light-microscopic studies on human cells in interphase, mitosis, and meiosis.

Electron micrographs reveal that the Ag-stainable substance is located on the outside of NOR's or around them but not in the chromosomes themselves. In association figures, the Ag-positive material lies between the acrocentric chromosomes. Light-microscopic studies show that the Ag stainability of the nucleolus in interphase is correlated with the function of the NOR, as seen from inactive and activated lymphocytes. Much more Ag-positive material is seen in prophase than in meta- and anaphase. It starts to increase again in late telophase. In male meiosis the NOR's remain Ag-positive until pachytene. First and second metaphase figures are negative. Experiments using RNase, TCA, and trypsin indicate that the Ag-stainable substance is an acidic protein. The precipitation of Ag granules in interphase nuclei seen in the electron microscope is greatest over the fibrillar component of the nucleolus. The most likely interpretation is that the Ag-stainable material is a component of ribonucleic protein accumulating around active NOR's. In mitosis some of this material remains at the NOR's. In first meiosis it is completely removed before diakinesis.

Animals↗
PubMed1980

Studies of mammalian chromosome replication. I. BrdU-induced differential staining patterns in interphase and metaphase chromosomes.

The patterns of differential staining based on the effects of BrdU-substitution in chromosomal DNA have been examined in both metaphase chromosomes and prematurely condensed chromosomes (PCC) of interphase Chinese hamster cells. Results indicate that differential staining may be obtained in chromosomes from all stages of the cell cycle and correspond to the semi-conservation mode of DNA replication. Such fidelity of differential staining in both interphase and metaphase chromosomes suggests that components essential for induction of differential staining are present throughout the cell cycle and chromosomes may contain similar structures and organization throughout the cycle.

Animals↗
PubMed1989

Molecular cytological differentiation of active from inactive X domains in interphase: implications for X chromosome inactivation.

A fluorescence in situ hybridization method using a biotinylated DNA probe specific for the centromeric region of the human X chromosome was used to differentiate the genetically active from the inactive X in interphase cells. With this technique, we were able to interpret both the relative position and the degree of condensation of the X chromosomes within the nucleus. We first established the specificity of fluorescence labelling of the hybridized probe by comparing its location and appearance (either dense or diffuse) when associated with a sex chromatin body (SCB) in early passage normal human female fibroblasts. In these cells, where the presence of inactive X chromatin was verified by identification of a 4',6-diamidino-2-phenyl indole (DAPI)-positive SCB in 85% of the cells examined, the X chromatin fluorescence was always associated with the SCB. The signal was dense in structure in 98% and peripheral in location in 80% of the nuclei. A second type of signal, diffuse in form, was observed in 85% of the nuclei and presumably represents the location of the active X chromosome. It was located peripherally or centrally with equal frequency and was not associated with any identifiable nuclear component. This diffuse signal was the major type associated with human male fibroblasts. In rodent x human hybrid cells containing a human inactive X, the fluorescent signal was associated with an SCB-like structure in only 13% of the nuclei; it was dense in 66% of the nuclei and equally peripheral or central in location. This indicates an alteration in the interphase structure of the human inactive X chromosome in hybrid cells which may explain its known instability with respect to genetic activity in such systems.

Animals↗
PubMed1992

Detection of cell-cycle stage by fluorescence in situ hybridization: its application in human interphase cytogenetics.

Distinct cell-cycle-dependent changes in the conformation of centromeric chromatin in a specific human chromosome containing alpha-satellite DNA have been demonstrated by fluorescence in situ hybridization (FISH). This method, based upon specific FISH signal morphology, allows simultaneous analysis of chromosomal aneuploidy and detection of specific cell-cycle stage(s) of human tumor and/or normal cell populations in a single preparation of interphase cells. This interphase cytogenetic procedure might prove useful for both basic and clinical research involving human cells.

Cell Cycle↗
PubMed1993

Ordering of probes surrounding the Ewing's sarcoma breakpoint on chromosome 22 using fluorescent in situ hybridization to interphase nuclei.

Eight probes were localized by fluorescent in situ hybridization to the region surrounding the Ewing's sarcoma breakpoint on chromosome 22. Three of these were initially ordered by pair-wise hybridization to metaphase chromosomes with differential detection of the probes. These and the remaining probes were then ordered by hybridizing two or three probes simultaneously to interphase nuclei. In the two probe experiments and some of the three probe experiments, the order was derived by comparing mean interphase distances between signals from the probes. In the three probe experiments, either two probes were detected with one fluorochrome and the third with another or all three probes were individually distinguished by detecting one probe with fluorescein isothiocyanate (FITC), one with Texas Red, and one with both fluorochromes to give a mixed color. The order of the signals was then noted. Greater than 60 percent of configurations with a discrete order were shown or deduced to be correct. These approaches are assessed and we demonstrate a more obvious predominating order when all three probes are differentially detected. The order of the probes was deduced to be centromere: D22S271: D22S260: lambda S1: D22S262: cosK1831: Ewing's sarcoma breakpoint: cosLIF: D22S261: lambda S15: telomere.

Cell Nucleus↗
PubMed1995

Meiotic segregation of the X and Y chromosomes and chromosome 1 analyzed by three-color FISH in human interphase spermatozoa.

Meiotic segregation of the X and Y chromosomes and chromosome 1 was analyzed by three-color fluorescence in situ hybridization (FISH) in 94,575 human interphase spermatozoa from four control subjects. More than 99% of the sperm cells were labeled. The proportions of X- and Y-bearing sperm were estimated to be 49.83% and 48.30%, respectively. The disomy rates were 0.04%, 0.009%, and 0.20% for the X and Y chromosomes and chromosome 1, respectively. Hyperhaploidy with an extra gonosome was found in 0.34% of spermatozoa, due to nondisjunction during meiosis I. The frequency of diploidy was 0.11% at meiosis I and 0.036% at meiosis II. Cohybridization of one autosomal and two gonosomal probes, in three-color FISH in interphase spermatozoa, seems to accurately discriminate diploidies from disomies, as well as the meiotic origin of gonosomal aneuploidies in sperm cells.

DNA Probes↗
PubMed1997

The monosomy 7 clone in interphase and metaphase cell population: a combined chromosome and primed in situ labeling study.

Loss of a chromosome 7 is associated with a poor prognosis in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Some studies have shown higher frequencies of monosomy 7 (-7) in dividing than nondividing myeloid cells, which might indicate that -7 confers a proliferative advantage on the host cell. As other groups have not been able to confirm this, we compared the -7 frequencies in bone marrow metaphases as studied with conventional cytogenetics and in interphase cells using primed in situ (PRINS) labeling. We found significantly higher -7 frequencies in metaphase than in interphase cells irrespective of diagnosis and presence or absence of additional chromosome aberrations. Further, we found a significant correlation between the -7 percentages in resting and dividing cells. Finally, as our material showed a clear male preponderance, Mitelman's Catalog of Chromosome Aberrations in Cancer was searched for -7. Of 815 cases with AML or MDS, 491 (60.3%) were found to be men. To our knowledge, this is the first observation of a clear deviation from the 1:1 sex ratio in -7 patients.

Acute Disease↗