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PubMed1995

FISH on metaphase and interphase chromosomes demonstrates the physical order of the genes for GPI, CRC, and LIPE in pigs.

The porcine genes for glucose phosphate isomerase (GPI), calcium release channel (CRC), and hormone-sensitive lipase (LIPE) map to the long arm of chromosome 6. Earlier studies, using fluorescence in situ hybridization (FISH), mapped the three loci to the same band, viz., 6q12. To ascertain the relative order of the three genes, we first conducted three double-color FISH experiments, cohybridizing two of the probes at a time. These experiments demonstrated that the gene order was cen-GPI-CRC-LIPE. This order was confirmed by cohybridizing the three probes to both metaphase and interphase chromosomes.

Animals↗

PubMed1996

M32, a murine homologue of Drosophila heterochromatin protein 1 (HP1), localises to euchromatin within interphase nuclei and is largely excluded from constitutive heterochromatin.

Mice possess two structural homologues of Drosophila HP1, termed M31 and M32 (Singh et al., 1991). We have previously shown that an M31-specific monoclonal antibody (MoAb), MAC 353, localises to constitutive heterochromatin (Wreggett et al., 1994). Here we report that a MoAb raised against the M32 protein (MAC 385) recognises a 22-kDa protein in murine nuclear extracts and that M32 is distributed in a fine-grain "speckled" pattern within interphase nuclei. M32 is also largely excluded from the large masses of constitutive heterochromatin that are labelled by MAC 353.

Animals↗

PubMed1997

Development of diagnostic tools for the analysis of 5p deletions using interphase FISH.

Cri-du-chat syndrome is associated with a deletion of the short arm of chromosome 5. Through the phenotypic and molecular analyses of individuals with a subset of the features associated with the syndrome, the genes involved in the syndrome have been mapped to two distinct critical regions. Deletion of a critical region in 5p15.2 results in the distinct facial features associated with the syndrome as well as the severe mental and developmental delay, while a deletion of 5p15.3 is associated only with the characteristic cat-like cry, the key diagnostic feature of the syndrome. Therefore, subtle differences in the extent of the 5p deletion can have a profound affect on the prognosis of the patient. In order to more easily differentiate between deletions that lead to the cri-du-chat syndrome phenotype and deletions that lead only to the isolated cat-like cry, we have constructed YAC contigs that span both critical regions. The YAC clones have been used to isolate cosmids mapping to each critical region and cosmids that lie just within the two critical region boundaries have been identified. We report here on the use of these cosmids as probes for fluorescent in situ hybridization experiments on interphase nuclei as a means of more accurately differentiating between small 5p deletions that coincide with a complete cri-du-chat syndrome phenotype and the severe mental and developmental delay that is associated with it and deletions that only delete the distal critical region that coincide with the isolated cat-like cry and a much improved prognosis.

Animals↗

PubMed2001

Detection of structural and numerical chromosome abnormalities in interphase cells using spectral imaging.

Chromosome abnormalities are common causes of congenital malformations and spontaneous abortions. They include structural abnormalities, polyploidy, trisomy, and mosaicism. In in vitro fertilization (IVF) programs, preimplantation genetic diagnosis (PGD) of oocytes and embryos has become the technique of choice to select against abnormal embryos before embryo transfer. For diagnosis of structural abnormalities, we developed case-specific breakpoint-spanning DNA probes. Screening of an in-house yeast artificial chromosome (YAC) library is facilitated by information from publicly available databases and published articles. Most numerical chromosome abnormalities, on the other hand, are detrimental to early embryonic development and increase with maternal age. We therefore developed a multichromosome screening technique based on spectral imaging to simultaneously detect and score as many as 10 different chromosome types. The probe set was chosen to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions. Detecting structural and numerical abnormalities in single interphase cells using spectral imaging is a powerful technique for multilocus genetic screening.

Chromosome Aberrations↗

PubMed2002

Suspension (S)-FISH, a new technique for interphase nuclei.

We describe a versatile method for performing fluorescence in situ hybridization (FISH) in suspension instead of on a slide as usually done. This so-called suspension-FISH (S-FISH) opens new possibilities for the analysis of shape and functions of the human interphase nucleus. The procedure is described and the first results using this approach are presented.

Cell Nucleus↗

PubMed1984

Distribution of calcium during interphase and mitosis as observed by ion microscopy.

The ion microscope, based on secondary ion mass spectrometry, has been used to demonstrate the distribution of calcium in the root tip cells of two plant species, Allium cepa and Vicia faba. Interphase nuclei showed higher intensities of calcium than cytoplasm, while nucleoli exhibited higher calcium intensities than the rest of the nucleoplasm. The chromosomes showed high intensities of calcium at all stages of mitosis. Calcium was also detected in the cell plate and phragmoplast region of dividing cells. It appears that during prophase calcium concentrates in the condensing chromosomes, and during telophase it is transferred to nucleoli. These observations suggest that chromosomes may serve as a reservoir of calcium during mitosis.

Calcium↗

PubMed1996

Methods for simultaneous interphase in situ hybridization and nuclear antigen immunocytochemistry in T47-D cells.

Procedures that combine immunocytochemistry (ICC) and in situ hybridization (ISH) techniques are now used to investigate phenotype/genotype relationships in the same cells. In this report we describe three rapid procedures for simultaneous detection of a nuclear antigen, progesterone receptors (PR), and the centromeric region of chromosome 11 (to which the human PR gene has been assigned) in T47-D cells. Proteins were stained by precipitates of horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) or alkaline phosphatase-nitroblue tetrazolium-X-phosphate (APase-NBT-X-Phosphate, blue color) respectively. To obtain a suitable contrast for the two labels, we detected DNA on PO-DAB and APase-NBT-X-phosphate-immunostained cells with interphasic fluorescent in situ hybridization (FISH). By contrast, we combined the APase-Fast Red ICC with an immunocytochemical ISH using alkaline phosphatase-NBT-X-phosphate detection. Only the procedure combining APase-NBT-X-phosphate ICC and FISH ensures optimal visualization of both the PR content and the number of chromosome 11. This method easily provides simultaneous localization of DNA and protein targets in the same cells and should be applicable to many other situations.

Antigens, Nuclear↗

PubMed2006

Simultaneous localization of MLL, AF4 and ENL genes in interphase nuclei by 3D-FISH: MLL translocation revisited.

BACKGROUND: Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. METHODS: Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. RESULTS: In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. CONCLUSION: The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.

Adolescent↗

PubMed1996

Detection of the t(14;18) chromosomal translocation by interphase cytogenetics with yeast-artificial-chromosome probes in follicular lymphoma and nonneoplastic lymphoproliferation.

PURPOSE: The aim of this study was to establish a fluorescence in situ hybridization (FISH) technique for the detection of t(14;18)(q32;q21), characteristic for follicular lymphoma (Kiel classification: centroblastic centrocytic [cb-cc] lymphoma). MATERIALS AND METHODS: After the FISH system had been established, parallel studies of lymph node biopsy specimens from 30 patients with cb-cc lymphoma and from 32 patients with nonneoplastic lymphoproliferation were performed by means of chromosome analysis, polymerase chain reaction (PCR), and FISH analysis. Two differently labeled yeast-artificial-chromosome (YAC) probes that contained the entire bcl-2 gene and the C-region of the immunoglobulin H (IgH) gene, respectively, were used to detect t(14;18) by FISH. RESULTS: The presence of the translocation is indicated by a red (Cy3)/green (fluorescien isothiocyanate [FITC]) double signal, which corresponds to the IgH/bcl-2 fusion gene, whereas in normal cells the signals are separate. Control studies showed that the double signal is visible in less than 1% of normal cells. FISH analysis was able to identify the t(14;18) in all cases of cb-cc lymphoma we studied. All bcl-2 breakpoints can be detected. Combined immunophenotyping and interphase cytogenetics demonstrated that t(14;18) was restricted to CD22+ B lymphocytes and never occurred in CD3+ T lymphocytes. In four of 32 cases of nonneoplastic lymphoproliferation, t(14;18) was also detected. CONCLUSION: FISH turned out to be the most sensitive method to detect t(14;18). Our FISH results confirm PCR data from other groups that found evidence for the presence of t(14;18) in nonneoplastic lymphoproliferation. It needs to be determined whether, in morphologically nonneoplastic processes, t(14;18) is associated with an increased risk for the development of non-Hodgkin's lymphoma.

Adult↗

PubMed2000

Treatment of leptomeningeal metastases evaluated by interphase cytogenetics.

PURPOSE: Although cytologic examination of CSF is the primary method for the evaluation of response to therapy for leptomeningeal metastases (LMMs), the procedure's sensitivity decreases throughout the course of protracted therapy. We studied whether this response could be monitored more accurately through the detection of numerical chromosomal aberrations by interphase cytogenetics, using fluorescence in situ hybridization (FISH). PATIENTS AND METHODS: Seven patients treated for LMMs and with a known numerical aberration for chromosome 1 in their pretreatment CSF were included in this study. Up to 16 consecutive CSF samples were analyzed by means of the fluorescence in situ hybridization (FISH) technique for cells with aberrant chromosome 1 content. The results of routine cytology and FISH analyses were compared and were correlated with each patient's neurologic status. RESULTS: Routine cytology detected malignancies in only 24 of the 76 samples, all of which were classified as chromosomally abnormal by FISH (except for two samples that could not be evaluated). Moreover, FISH demonstrated aneusomic cells in 32 additional samples, which could therefore be classified as malignant. The FISH results correlated better with patient neurologic status in that more malignant cells were detected in the CSF of neurologically deteriorating patients. CONCLUSION: Using FISH in addition to performing routine cytologic examination of CSF led to a more accurate evaluation of response to treatment in patients treated for LMMs.

Antineoplastic Agents↗

PubMed1997

Developmentally regulated activation of apoptosis early in Xenopus gastrulation results in cyclin A degradation during interphase of the cell cycle.

Previous work identified a developmental timer that controls the stability of cyclin A protein in interphase-arrested Xenopus embryos. It was shown that cyclins A1 and A2 abruptly become unstable in hydroxyurea-treated embryos at the time that untreated embryos are beginning gastrulation (early gastrulation transition; EGT). We have demonstrated here that cyclins A1 and A2 are degraded at the equivalent of the EGT by the ICE-like caspases that are responsible for programmed cell death or apoptosis. Analysis of embryos treated with hydroxyurea or cycloheximide showed widespread cellular apoptosis coincident with cyclin A cleavage. Our data further indicate that the apoptotic pathway is present in Xenopus embryos prior to the EGT; however, it is maintained in an inactive state in early cleaving embryos by maternally encoded inhibitors. Characterization of the timing of the activation of apoptosis implicates the initiation of zygotic transcription at the mid-blastula transition (MBT) in the suppression of apoptosis in normal embryos. The decreased biosynthetic capacity of embryos treated with hydroxyurea or cycloheximide most likely interferes with the ability to maintain sufficient levels of apoptotic inhibitors and results in widespread apoptosis. Our results suggest a scenario whereby the apoptotic pathway is suppressed in the early cleaving embryo by maternally contributed inhibitors. Degradation at the EGT of maternal RNAs encoding these inhibitors is compensated for by new zygotic transcription beginning at the MBT. This indicates that the interval between the MBT and the EGT represents a critical developmental period during which the regulation of embryonic cellular processes is transferred from maternal to zygotic control.

Animals↗

PubMed2003

Interphase and monoastral-mitotic phenotypes of overexpressed MAP4 are modulated by free tubulin concentrations.

The microtubule-associated protein 4 (MAP4) has recently been shown to counteract destabilization of interphase microtubules caused by catastrophe promotion but not by tubulin sequestering. To address how MAP4 discriminates between destabilization of microtubules by these two mechanisms, we have evaluated the combined phenotypes of MAP4 coexpressed with Op18/stathmin family member derivatives with either catastrophe-promoting or sequestering activities. This approach relies on the finding that overexpression of MAP4 alone stabilizes microtubules during all phases of the cell cycle in human leukemia cells, and causes a potent mitotic block and a dramatic, previously unobserved, phenotype characterized by large monoastral spindles. Coexpression of either catastrophe-promoting or tubulin-sequestration-specific Op18 derivatives was found to modulate the activity of ectopic MAP4 during mitosis, but with differential functional outcome. Interestingly, the tubulin-sequestering derivative suppressed the monoastral mitotic phenotype of MAP4 (i.e. coexpression facilitated the formation of functional spindles). To evaluate whether this phenotypic suppression could be explained by tubulin-sequestration-dependent modulation of MAP4 activity, a plasma-membrane-targeted, tubulin-sequestering chimera was constructed to decrease the cytosolic free tubulin concentration substantially. This chimera likewise suppressed the monoastral phenotype caused by overexpression of MAP4, suggesting a direct downregulation of MAP4 activity by reduced free tubulin concentrations.

Flow Cytometry↗

PubMed2004

Trichostatin A-induced histone acetylation causes decondensation of interphase chromatin.

The effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a reversible decondensation of dense chromatin regions and led to a more homogeneous distribution. These structural changes were quantified by image correlation spectroscopy and by spatially resolved scaling analysis. The image analysis revealed that a chromatin reorganization on a length scale from 200 nm to >1 microm was induced consistent with the opening of condensed chromatin domains containing several Mb of DNA. The observed conformation changes could be assigned to the folding of chromatin during G1 phase by characterizing the effect of TSA on cell cycle progression and developing a protocol that allowed the identification of G1 phase cells on microscope coverslips. An analysis by flow cytometry showed that the addition of TSA led to a significant arrest of cells in S phase and induced apoptosis. The concentration dependence of both processes was studied.

Acetylation↗

PubMed2004

Protein 4.1R regulates interphase microtubule organization at the centrosome.

In human red blood cells, protein 4.1 (4.1R) stabilizes the spectrin-actin network and anchors it to the plasma membrane. To contribute to the characterization of functional roles of 4.1R in nonerythroid cells, we analysed the effect of ectopic expression of 4.1R isoforms on interphase microtubules in fibroblastic cells. We found that specific 4.1R isoforms disturbed the microtubule architecture but not the actin cytoskeleton. Biochemical sedimentation and/or confocal microscopy analyses showed that the pericentriolar components gamma-tubulin and pericentrin remained at centrosomes, whereas the distributions of proteins p150Glued and the dynein intermediate chain were altered. Remarkably, 4.1R was displaced from the centrosome. In microtubule depolymerizing-repolymerizing assays, 4.1R-transfected cells showed an ability to depolymerize and nucleate microtubules that was similar to that of untransfected cells; however, microtubules became disorganized soon after regrowth. In microtubule-depolymerized transfected cells and during the initial steps of microtubule regrowth, centrosomal 4.1R localized with gamma-tubulin but did not when microtubules became disorganized. To learn more about centrosomal 4.1R function, isolated centrosomes were examined by confocal microscopy, western blot and in vitro microtubule aster-assembly assays. The experiments showed that 4.1R was present in isolated centrosome preparations, that it remained in the center of in-vitro-assembled microtubule asters and that more asters were assembled by the addition of protein 4.1R fused to glutathione-S-transferase. Together, these results indicate that 4.1R plays a key role at the centrosome, contributing to the maintenance of a radial microtubule organization.

Actins↗

PubMed2006

Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23.

B23 (or nucleophosmin, NPM) is a multifunctional protein involved in ribosome biogenesis, control of centrosome duplication and in sensing cellular stress. It is phosphorylated during interphase by casein kinase 2 (CK2) and during mitosis by cyclin-dependent kinase (CDK). In this study we have addressed the role of these phosphorylation events in the dynamics and location of protein B23. Mutation of the CK2 phosphorylation site to alanine results in slower recovery of the mutant compared with the wild-type protein as measured by fluorescence recovery after photobleaching (FRAP). Immunofluorescence studies using an antibody against phosphorylated Thr199 revealed that B23 is phosphorylated at this CDK1 site at the start of mitosis and is dephosphorylated during anaphase. The CDK1-type phosphorylation sites are in the nucleic acid binding region of B23 and may contribute to its dissociation from the nucleolus during mitosis. A Thr to Glu mutant of the CDK1-type sites as well as other members of the nucleoplasmin family that lack the C-terminal nucleic-acid-binding region showed a greater mobility and/or faster recovery than wild-type B23.1, the longer variant. These results provide evidence that phosphorylation at these sites reduces the affinity of B23 for nucleolar components and might be a factor in regulating its location during the cell cycle.

Amino Acid Sequence↗

PubMed1992

Different patterns of rDNA organization at interphase in nuclei of wheat and rye.

The physical location of the rDNA repeating units (25 S, 18 S and 5.8 S rRNA genes and the intergenic spacer sequences) was investigated in rye (Secale cereale L.) and wheat (Triticum aestivum L.) root tip meristematic cells by in situ hybridization using light and electron microscopy. The rDNA sequences are organized differently in the two related and intercrossable species. In rye (2n = 14, one pair of chromosomes with nucleolar organizing regions, NORs), two condensed blocks of rDNA-containing chromatin occurred in each interphase nucleus. The blocks were associated with the periphery of nucleoli and a single-labelled, decondensed rDNA fibre extended into the nucleolus from the block. We term this expression pattern terminal decondensation. In wheat (2n = 6x = 42, five pairs of chromosomes with NORs), inactive condensed labelled chromatin was found unassociated with nucleoli. Active NORs had some condensed rDNA associated with the nucleolar periphery, but, in contrast to rye, condensed rDNA was also found within the nucleolus. The condensed labelled rDNA in wheat nucleoli was visible as fluorescent foci in the light microscope and labelled condensed chromatin in the electron microscope. Its absence in rye shows that condensed rDNA need not be present in active plant nucleoli. Diffuse labelled sites of rDNA, likely to represent actively transcribed rDNA, were found in both rye and wheat. Active rDNA loci in wheat have many expressed segments separated by unexpressed, condensed, rDNA-fragmented decondensation-while each locus in rye has a single, unexpressed perinucleolar condensed block of rRNA genes. Thus the positions of actively transcribed genes within the tandem arrays of rDNA at each locus are fundamentally different in the two cereals. The NOR chromosome appeared to extend through the nucleolus, and active rDNA sequences did not loop out from chromatin into the nucleolus as is frequently described in nucleolar models.

Cell Nucleolus↗

PubMed1992

Cyclin A-cdc2 kinase does not trigger but delays cyclin degradation in interphase extracts of amphibian eggs.

Purified cyclin B-cdc2 kinase has been shown previously to trigger cyclin degradation in interphase frog extracts by initiating a cascade of reactions that includes cyclin ubiquitinylation and ends with proteolysis. However, cyclin A-cdc2 kinase was not assayed in these early experiments. Here we have shown that full-length recombinant human cyclin A failed to induce cyclin degradation when it was added to frog extracts free of cyclin B, although it formed an active kinase complex with Xenopus cdc2. A highly purified kinase complex containing a truncated human cyclin A and starfish cdc2 also failed to switch on the cyclin degradation pathway. In contrast, both recombinant cyclin B and highly purified cyclin B-cdc2 kinase readily triggered degradation of both cyclins B and A in frog extracts. Whilst free cyclin A had no inhibitory effect, cyclin A-cdc2 kinase delayed degradation of both cyclins A and B induced by cyclin B-cdc2 kinase. The finding that cyclin A-cdc2 kinase cannot turn on, and even delays, cyclin destruction may be essential to prevent premature inactivation of MPF (maturation-promoting factor) before complete condensation of chromosomes and formation of the metaphase spindle.

Animals↗

PubMed1993

Distribution of snRNPs, splicing factor SC-35 and actin in interphase nuclei: immunocytochemical evidence for differential distribution during changes in functional states.

Small nuclear ribonucleoproteins (snRNPs) play an integral role in the processing of pre-mRNA in eukaryotic nuclei. snRNPs often occur in a speckled intranuclear distribution, together with the non-snRNP splicing factor SC-35. snRNPs have also been shown to be associated with actin in the nuclear matrix, suggesting that both actin and snRNPs may be involved in the processing and transport of transcripts. The work reported here was undertaken to compare the spatial relationship of snRNPs, SC-35, and intranuclear actin in neuronal and non-neuronal cell types. In undifferentiated PC12 cells and in non-neuronal cells growing in association with dorsal root ganglion neurons, confocal immunocytochemistry revealed a typical, speckled distribution of snRNP aggregates, which colocalized with the SC-35 splicing factor. In contrast, a unique snRNP distribution was observed in dorsal root ganglion neurons in vitro and in PC12 cells differentiated by nerve growth factor. In nuclei of these cells, snRNPs were predominantly located at the periphery where they formed a spherical shell apposed to the nuclear envelope. Ultrastructural immunogold labelling of snRNPs in dorsal root ganglion neurons in vitro confirmed this distribution. In contrast, SC-35 remained distributed in a speckled pattern throughout nuclei of dorsal root ganglion neurons and PC12 cells, even in cases where snRNPs were almost exclusively positioned at the nuclear periphery. In non-neuronal cells in dorsal root ganglion cultures and in undifferentiated PC12 cells, snRNP aggregates were frequently associated with actin aggregates, as determined by Nearest Neighbor Analyses. In PC12 cells, this spatial relationship was altered during nerve growth factor-induced differentiation, prior to the time at which these cells showed morphological evidence of differentiation. Specifically, Nearest Neighbor Analyses between snRNP and actin aggregates in PC12 cells exposed to nerve growth factor for 4 hours revealed that snRNP and actin aggregates exhibited a closer association than in undifferentiated cells. These results suggest that sites of pre-mRNA processing and transcription may differ between cell types, and that the functions of snRNPs and actin within interphase nuclei may be related. The results also indicate that the distribution of snRNPs is dynamic and that it may depend upon the functional state of the cell as well as upon its state of differentiation.

Actins↗