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PubMed1994

Ultrastructural localization of filamentous actin within neuronal interphase nuclei in situ.

Previous biochemical studies utilizing isolated nuclei and nuclear matrices have shown actin to be a constituent of the interphase nucleus. In addition, recent ultrastructural work has shown the presence of actin and myosin within nuclei of interphase cells in situ. It was unclear, however, whether this intranuclear actin is present in the unpolymerized globular actin or the filamentous (F)-actin form. The present work, using confocal microscopy and ultrastructural cytochemical techniques, demonstrates the presence of F-actin within interphase nuclei of intact, uncompromised, dorsal root ganglion neurons in vitro and in vivo. Labeling by FITC-phalloidin detected the presence of intranuclear F-actin adjacent to the nucleolar periphery in a small fraction of cells in vitro, an observation confirmed by three-dimensional reconstruction. Ultrastructural analyses of cells exposed to heavy meromyosin (HMM), showed the presence of typical "arrowhead" complexes. The observation that these complexes were associated with nucleoli confirms that the intranuclear ligand detected by FITC-phalloidin indeed represents F-actin. Postembedding labeling with HMM conjugated to 20-nm colloidal gold (HMM-Au20) resulted in labeling similar to that obtained with HMM. However, HMM-Au20 was found to label a much larger fraction of cells, both in vitro and in vivo, than did FITC-phalloidin or HMM. This finding indicates that labeling with HMM-Au20 more accurately reflects the extent of actin polymerization in nuclei. Results from double labeling with HMM-Au20 and an antibody to alpha-sarcomeric actin confirmed that only a small amount of nuclear actin is in the F-form. Together, these results represent a first ultrastructural demonstration of the presence of F-actin in nuclei of neurons. While the role of nuclear F-actin has yet to be defined, the results suggest that F-actin may represent a component of the molecular motor responsible for the dynamic positioning of specific chromatin domains into the tissue-specific, nonrandom patterns observed in many cell types.

Actins↗
PubMed1995

Nonrandom arrangement of bovine satellite I DNA within the interphase nucleus of Madin-Darby bovine kidney cells.

The distribution of bovine satellite DNA was examined in Madin-Darby bovine kidney cells for each hour of interphase. Cells were grown on coverslips and probed using biotinylated cloned fragments of bovine satellite I DNA. Hybridization was detected using a streptavidin-alkaline phosphatase conjugate. Cells were projected onto a grid of fixed dimensions and the distribution of hybridization signal was recorded. A chi 2 analysis of fit compared this distribution of signals to a random distribution generated from the poisson distribution. Nuclear localization of bovine satellite I DNA was found to be nonrandom throughout interphase except at the G1/S border and S = 6 h. Moreover, distinct patterns of hybridization were also observed at specific times during interphase.

Animals↗
PubMed1995

Influence of transcription and replication on the in situ resolution of immunoglobulin heavy-chain constant region genes: an interphase cytogenetics analysis.

An interphase cytogenetics analysis was performed to investigate whether replication and transcription could influence in situ resolution of immunoglobulin (Ig) heavy chain constant region genes. A plasmid probe recognizing five C gamma segments separated by known linear DNA distances was hybridized in situ and visualized by digital fluorescence microscopy. In interphase nuclei from phytohemagglutinin (PHA)-stimulated lymphocytes, the gamma genes were resolved as one to three signals per allele in the majority of nuclei, whereas in a minority, complex patterns of several signals per allele could be observed. The latter were restricted to nuclei in an early stage of the S phase, as assessed by hybridization experiments performed in cells grown in the presence of bromodeoxyuridine. To investigate whether the in situ resolution of the C gamma segments could vary as a function of the transcription activity of the locus, the C gamma probe was subsequently hybridized to nuclei from a mature B cell line (JVM-2), which produces gamma transcripts as shown by in situ RNA hybridization experiments. Primary human fibroblasts were further used as representative of a non-lymphoid cell type with transcriptionally inactive Ig genes. When Gl nuclei from the three cell types were compared in terms of the in situ resolution of the C gamma locus, JVM-2 cells were found to include the highest percentage of higher resolution patterns (three to five signals per allele in 28% of nuclei), fibroblasts the lowest (three signals per allele, 2%), while PHA-stimulated lymphocytes occupied an intermediate position between the other two cell types (three or four signals per allele, 15%). The data show that the in situ resolution of Ig C gamma genes varies throughout the cell cycle and is influenced by the transcriptional activity of the locus. The variability of the resolution patterns observed appears to reflect different levels of chromatin packaging, which in turn are likely to influence the probe accessibility to its target. These observations are relevant for the interpretation of data from interphase cytogenetics analysis of independent, but closely spaced, DNA segments.

Cells, Cultured↗
PubMed1991

Relative order determination of four Yp cosmids on metaphase and interphase chromosomes by two-color competitive in situ hybridization.

Two-color competitive in situ hybridization was used to cytogenetically order four Yp cosmid probes, located in the pseudo-autosomal and TDF regions. The probes were hybridized by pairs to metaphase and interphase chromosomes. On metaphase chromosomes, determination of order between sequences separated by 3 Mb from each other was possible on a statistical basis, whereas the relative position of sequences 0.6 Mb apart could not be determined. On interphase chromosomes the complete order between sequences separated by 0.6-6 Mb was obtained rapidly by measuring the distances between two cosmid spots of every cosmid pair used in 28 to 60 nuclei. Results demonstrate the potential power of fluorescent in situ hybridization at interphase for high resolution cosmid mapping.

Binding, Competitive↗
PubMed1991

In situ hybridization as a rapid means to assess meiotic pairing and detection of alien DNA transfers in interphase cells of wide crosses involving wheat and rye.

The objectives of this study were to determine if biotin-labelled total genomic DNA of rye (Secale cereale L.) could be used to (i) preferentially label rye meiotic chromosomes in triticale and (ii) detect translocation stocks at interphase and/or early prophase by in situ hybridization. Welsh triticale, a wheat-rye segmental amphiploid, and Kavkaz wheat, a wheat-rye translocation were used. The results indicated that labelled chromosomes of rye and unlabelled chromosomes of wheat could be observed throughout all meiotic stages in the triticale. For Kavkaz wheat, the presence of the translocated 1RS chromosome arm of rye was detected at the interphase or very early prophase stage. Rapid assessment of feasibility of gene transfers and detection of alien DNA in somatic cells at the interphase stage by in situ hybridization allows for rapid decision-making and saves time and expense in plant breeding programs.

Chromosomes↗
PubMed1987

Do individual allocyclic chromosomes in metaphase reflect their interphase domains?

Individual S phase allocyclic chromosomes have been analyzed in Bloom syndrome lymphocytes, in cells with an r(9), and in hypotetraploid Ehrlich mouse ascites cells treated with 1-methyl-2-benzyl hydrazine. On the basis of the following observations, we conclude that such chromosomes more or less reflect their domains in interphase: (1) The S phase allocyclic chromosomes have the same structure as S phase prematurely condensed chromatin (PCC) in fused cells; in other words they form limited areas of chromatin dots; (2) the allocyclic chromosome is the only chromosome in a metaphase plate which synthesizes DNA simultaneously with interphase nuclei; (3) the size of the allocyclic chromosomes is related to the size of the corresponding metaphase chromosome; and (4) the S phase allocyclic chromosomes resemble closely the chromosome domains in interphase made visible with biotinylated human DNA. A variety of evidence shows that most allocyclic chromosomes are simply left behind in their cycle, which presumably is caused by a deletion or inactivation of a hypothetical coiling center situated on each chromosome arm.

Animals↗
PubMed1989

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro.

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126 +/- 36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 micron thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20-30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.

Animals↗
PubMed1984

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes.

Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (Xqter----Xp22.2::Yq11----Yqter) contained in the Chinese hamster X human hybrid cell line 445 X 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.

Cell Nucleus↗
PubMed1985

New data on the in-situ position of the inactive X chromosome in the interphase nucleus of human fibroblasts.

The in situ spatial distribution of nucleolus-organizing-region (NOR) bearing chromosomes in relation to the inactive X chromosome was studied during interphase in human fibroblasts. The respective positions of these chromosomes were examined in 30 growing and 32 resting fibroblasts from reconstructed nuclei, using nucleoli and the Barr body as ultrastructural markers. Experimental values for the distance between the nucleoli and the Barr body were estimated by their coefficient of closeness and compared to the uniform distribution. The following results were obtained: the distribution patterns for the two populations of nuclei were similar, the distribution of the NOR-bearing chromosomes in relation to the inactive X chromosome varied and differed significantly from a uniform distribution, and in many cases the Barr body was observed to be in a juxta-nucleolar position. The internal distribution revealed by this study is compared with the data in the literature, especially with the conflicting data obtained by other methods used to determine the interphase arrangement of chromosomes. The relationship between interphase and metaphase arrangements such as can be deduced with these methods, is discussed in relation to the mechanisms of the formation of metaphase plates or chromatid translocations.

Cell Nucleolus↗
PubMed1982

Specific staining of 9h in human somatic interphase cells by D 287/170.

Somatic interphase cells from males and females with normal karyotypes and with variants of the heterochromatic regions on chromosomes 9 and Y were stained with the fluorochrome D 287/170. The results showed that only 9h retained the ability to stain with D 287/170 in the interphase state, whereas 15ph and Yqh lost the specific staining pattern seen in metaphase. The number and size of the specific stained interphase bodies correlated with the ploidy of the cell population and the size of the 9h as seen in metaphase.

Chromosomes, Human, 6-12 and X↗
PubMed1995

The influence of B chromosomes on rDNA organization in rye interphase nuclei.

Patterns of rye rDNA organization in interphase nuclei were studied through the use of in situ hybridization in spreads of root meristem cells from plants with and without B chromosomes (Bs). In cells from plants without Bs each rDNA locus is organized as a single perinucleolar knob of condensed chromatin with decondensed chromatin inside the nucleolus. In plants with Bs there is a marked modification of the pattern, found in more than 23% of nuclei, which involves several regions of condensed chromatin interspersed with decondensed chromatin inside the nucleolus. This B-induced alteration in rDNA interphase organization suggests a change in expression of the rRNA genes located on the A chromosomes probably related to the reduction in nuclear RNA observed previously in plants with Bs. The influence of the Bs on the expression of A chromosome genes, through rearrangement of interphase chromatin, could provide the basis of an explanation for some of the known phenotypic effects of B chromosomes in rye.

Cell Nucleus↗
PubMed1984

Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei.

Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver. In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes. In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules. In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes. In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes. In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components. The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.

Acetylation↗
PubMed1988

Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries.

A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzyme-labeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laser-scanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.

Chromosomes, Human↗
PubMed1982

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX.

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.

Alcian Blue↗
PubMed1999

Third-strand in situ hybridization (TISH) to non-denatured metaphase spreads and interphase nuclei.

A methodology has been developed for binding oligodeoxyribonucleotide 'third strands' to duplex DNA targets in fixed but not additionally denatured metaphase spreads and interphase nuclei under conditions found to be optimal in solution. Third-strand in situ hybridization (TISH) at pH 6.0 of a psoralen- and biotin-modified 16-nucleotide homopyrimidine third strand to a unique multicopy target sequence in human chromosome 17 alpha-satellite (D17Z1 locus) is described. UVA-photofixed third strands, rendered fluorescent by fluorescein isothiocyanate-labeled avidin, are reproducibly centromere-specific for chromosome 17, and visible without amplification of the signal in lymphocyte and somatic cell hybrid spreads and interphase nuclei. Two third-strand-specific D17Z1 haplotypes were identified. TISH has potential diagnostic, biochemical, and flow cytometric applicability to native metaphase and interphase chromatin.

Base Sequence↗
PubMedDate not supplied

Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH).

Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as "interphase cytogenetics", can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed.

Cytological Techniques↗
PubMedDate not supplied

Interphase cytogenetics and comparative genomic hybridization of human epithelial cancers and precursor lesions.

The accuracy of cytogenetic analyses of human solid cancers has improved enormously over the past decade by the introduction and refinement of DNA in situ hybridization (ISH) techniques. This methodology can be applied to cells in the interphase state, thereby making it an excellent tool for the delineation of chromosomal aberrations in solid tumors. The use of non-isotopic ISH to intact and disaggregated cancer specimens will be discussed, as well as comparative genomic hybridization (CGH) with tumor-derived DNAs. In this review we will focus on hybridocytochemical interphase approaches for the detection of chromosomal changes in frequently occurring human epithelial malignancies, e.g., breast, lung, and prostate carcinomas. We will further discuss the use of ISH procedures for the genetic analysis of precursor conditions leading to invasive carcinomas. Knowledge concerning these precancerous conditions is increasing, and its importance in cancer prevention has been recognized. Interphase cytogenetics by ISH, as well as CGH, with DNAs derived from microdissected, precancerous, dysplastic tissue areas will increase our understanding of these lesions, both at the investigative and diagnostic levels.

Humans↗