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Results for “chromatin organization”

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At least 19 recordsLinked to original sources

PubMed1976

Chromatin organization in nuclei of sea urchin embryos. Comparison with the chromatin organization of the sperm.

The chromatin in sea urchin embryo nuclei and that in sperm heads are both organized in nucleosomes but show marked differences when analyzed by endonuclease digestion. Sperm chromatin DNA appears to be totally organized in nucleosomes that are highly resistant to nuclease hydrolysis. The kinetics of formation of acid-soluble oligonucleotides is slow and concerns only about 50% of the total DNA. In contrast, the DNA of embryo chromatin does not appear to be totally organized in nucleosomes since 5 to 10% is rapidly and preferentially hydrolysed into acid-soluble oligonucleotides without any appreciable fragmentation of the remaining parts. Futher digestion causes the formation of the usual pattern of DNA bands, as detected by gel electrophoresis. The length of the DNA segment associated with the embryo nucleosomes appears to be shorter than that of the DNA segment associated with the sperm nucleosomes. The kinetics of formation of acid-soluble oligonucleotides upon digestion of embryo chromatin is much faster than that of sperm chromatin and concerns almost all the chromatin DNA.

Animals↗

PubMed1996

In vitro dynamics of chromatin organization and migration.

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.

Animals↗

PubMed1995

Chromatin organization during mouse oocyte growth.

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I-III, 10-40 microns in diameter: 100% NSN vs. 0% SN; class VII 70-80 microns in diameter: 47.3% NSN vs. 52.3 SN, in 4-6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation.

Animals↗

PubMed2006

A high mobility group protein binds to long CAG repeat tracts and establishes their chromatin organization in Saccharomyces cerevisiae.

Long CAG repeat tracts cause human hereditary neurodegenerative diseases and have a propensity to expand during parental passage. Unusual physical properties of CAG repeat tracts are thought to contribute to their instability. We investigated whether their unusual properties alter the organization of CAG repeat tract chromatin. We report that CAG repeat tracts, embedded in yeast chromosomes, have a noncanonical chromatin organization. Digestion of chromatin with the restriction enzyme Fnu4HI reveals hypersensitive sites occurring approximately 125 bp apart in the repeat tract. To determine whether a non-histone protein establishes this pattern, we performed a yeast one-hybrid screen using CAG repeat tracts embedded in front of two reporter genes. The screen identified the high mobility group box protein Hmo1. Chromatin immunoprecipitation of epitope-tagged Hmo1 selectively precipitates CAG repeat tracts DNAs that range from 26 to 126 repeat units. Moreover, deletion of HMO1 drastically alters the Fnu4HI digestion pattern of CAG repeat chromatin. These results show that Hmo1 binds to CAG repeat tracts in vivo and establish the basis of their novel chromatin organization.

Base Sequence↗

PubMed1981

Macronuclear chromatin organization in Paramecium primaurelia.

Chromatin organization and transcriptional activity in the macronucleus of the ciliate Paramecium primaurelia have been studied by electron microscopic examination of spread chromatin preparations. Under spreading conditions which inhibit the activity of endogenous nucleases and proteases, various morphologically distinct types of chromatin are seen: (1) Largely condensed chromatin in the form of 30-40 nm supranucleosomal globules which aggregate to form knobbly 40-60 nm fibres and unravel into 12 nm nucleosomal filaments; (2) In rapidly growing cells, extended chromatin arranged groups of 4-8 adjacent nucleofilaments, each bearing numerous and very densely packed lateral fibrils. These fibrils are 20 nm thick, have a smooth outline and extend to lengths of up to 3 micron. Such transcripts are grouped in units along the chromatin axis and are apparently organized in register in adjacent filaments; (3) In starved cells, extended nucleofilaments bearing only occasional RNP fibrils. These various morphological characteristics are discussed in terms of macronuclear transcriptional activity and the polyploid nature of macronuclei.

Animals↗

PubMed1991

Chromatin organization in Entamoeba histolytica.

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.

Actins↗

PubMed1981

Microfluorometric investigations of chromatin structure. I. Evaluation of nine DNA-specific fluorochromes as probes of chromatin organization.

The ability of the highly condensed chromatin of small thymocyte nuclei and the more loosely organized chromatin of hepatocyte nuclei to interact with nine DNA-specific fluorochromes was assessed by microfluorometry. Although the results obtained with five of the fluorochromes - mithramycin, 7-aminoactinomycin D, Hoechst 33258, DAPI, and propidium iodide - were found to be virtually unaffected by differences in the degree of condensation of the chromatin, the values obtained with the remaining fluorochromes - proflavine, quinacrine mustard, berberine sulfate, and pyronin Y - appeared to be affected significantly by organizational differences of the chromatin. All of the latter "structural probes," except quinacrine mustard, produced fluorescence values which were higher in the 2c nuclei of hepatocytes than in the nuclei of small thymocytes. Quinacrine mustard yielded higher values in thymocyte nuclei; and in the hepatocyte polyploid series (2, 4, and 8c), it did not produce the expected multiples of the 2c value. Pretreatment of the two types of nuclei with RNase affected their total fluorescence in unpredictable ways. While RNase extraction lessened the differences between thymocyte and 2c hepatocyte nuclei stained with propidium iodide, Hoechst 33258, proflavine, and berberine sulfate, it increased the differences between nuclei stained with mithramycin, quinacrine mustard, pyronin Y, and 7-aminoactinomycin D. The ability of RNA-depleted chromatin to interact with various types of fluorochromes might be a useful parameter in subsequent studies of chromatin organization.

Animals↗

PubMed2000

The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome.

In yeast alpha cells the a cell-specific genes STE6 and BAR1 are packaged as gene-sized chromatin domains of positioned nucleosomes. Organized chromatin depends on Tup1p, a corepressor that interacts with the N-terminal regions of H3 and H4. If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes. Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region. To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed. After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor. In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome. These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains.

Chromatin↗

PubMed1985

Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.

Chromatin↗

PubMed1995

Genetic and physical interactions between yeast RGR1 and SIN4 in chromatin organization and transcriptional regulation.

The SIN4 and RGR1 genes of Saccharomyces cerevisiae were identified by mutations in quite different genetic screens. We have shown that the SIN4 gene product is required for proper transcriptional regulation of many genes and that a sin4 mutation can affect either activation or repression of specific genes. We have suggested that this dual nature of SIN4 in transcriptional regulation is due to its involvement in chromatin organization. We now report that the role of RGR1 in gene regulation is similar to that of SIN4. SIN4 and RGR1 both function as negative transcriptional regulators of HO and IME1, and mutations in either gene lead to decreased expression of other genes including CTS1. Strains with sin4 or rgr1 mutations both have phenotypes similar to those caused by histone mutations, including suppression of delta insertion into promoters (Spt- phenotype), activation of promoters lacking UAS elements, and decreased superhelical density of plasmid DNA molecules. Overexpression of RGR1 suppresses the temperature sensitivity due to a sin4 mutation. Finally, we use yeast strains expressing GST fusion proteins to demonstrate that the Sin4p and Rgr1p proteins are physically associated in vivo. These results indicate that Sin4p and Rgr1p act together in vivo to organize chromatin structure and thus regulate transcription.

Chromatin↗

PubMed2025

ChromaFactor: Deconvolution of single-molecule chromatin organization with non-negative matrix factorization.

The investigation of chromatin organization in single cells holds great promise for identifying causal relationships between genome structure and function. However, analysis of single-molecule data is hampered by extreme yet inherent heterogeneity, making it challenging to determine the contributions of individual chromatin fibers to bulk trends. To address this challenge, we propose ChromaFactor, a novel computational approach based on non-negative matrix factorization that deconvolves single-molecule chromatin organization datasets into their most salient primary components. ChromaFactor provides the ability to identify trends accounting for the maximum variance in the dataset while simultaneously describing the contribution of individual molecules to each component. Applying our approach to two single-molecule imaging datasets across different genomic scales, we find that these primary components demonstrate significant correlation with key functional phenotypes, including active transcription, enhancer-promoter distance, and genomic compartment. Also, we find that some bulk trends exist at the single-cell level, but only in a small fraction of cells, suggesting that critical changes in genome organization may be driven by specific rare subpopulations rather than occurring uniformly across all cells. ChromaFactor offers a robust tool for understanding the complex interplay between chromatin structure and function on individual DNA molecules, pinpointing which subpopulations drive functional changes and fostering new insights into cellular heterogeneity and its implications for bulk genomic phenomena.

Animals↗

PubMed1994

ABFI contributes to the chromatin organization of Saccharomyces cerevisiae ARS1 B-domain.

The involvement of the ABFI transcription factor in organizing the chromatin structure of the Saccharomyces cerevisiae ARS1 region has been previously postulated. We studied the ARS1 chromatin structure both on the chromosome and on plasmids carrying wild type or mutated ABFI binding sites, using a recently developed no-background technique for nucleosome mapping, coupled with high resolution micrococcal nuclease in vivo footprinting. We show that ABFI protein acts as a boundary element of chromatin structure, by limiting the invasion by nucleosomes toward the essential A-domain.

Base Sequence↗

PubMed1981

Chromatin organization in the rat hypothalamus during early development.

The organization of chromatin in neuronal and glial nuclei isolated from different brain regions of rats during development was studied by digestion of nuclei with micrococcal nuclease. A short chromatin repeat length (approx. 176 base-pairs compared with that of glial nuclei from foetal cerebral cortex (approx. 200 base-pairs) was present in hypothalamic neurons throughout the ages studied, which was similar to the repeat length of cortical neurons from 7- and 25-day-old animals (approx. 174 base-pairs). Whereas in cortical neurons the chromatin repeat length shortened from approx. 200 base-pairs in the foetus to approx. 174 base-pairs in the first postnatal week, the short chromatin repeat length of hypothalamic neurons was already present 2 days before birth, indicating that hypothalamic neurons differentiate earlier than cortical neurons during brain development.

Animals↗

PubMed1978

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication.

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature-sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation.

Cell Count↗

PubMed1983

Homogeneously staining regions and double minutes in a human cell line: chromatin organization and DNA content.

Homogeneously staining regions (HSR) and double minutes, two novel chromosome abnormalities, are believed to represent sites of gene amplification. The basic organization of chromatin in an HSR in a human neuroblastoma cell line was shown, by electron microscopy, to be similar to that in a normal chromosome. Double minutes from another human neuroblastoma cell line were found, by dry mass determination, to contain an average of 1.41 X 10(-14) g DNA. This quantity corresponds to 6.38 X 10(3) kilobase DNA per double minute.

Cell Line↗

PubMed1999

Gene expression and chromatin organization during mouse oocyte growth.

Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis.

Age Factors↗

PubMed1985

Nuclear volume and chromatin organization in some radiosensitive and radioresistant mammalian cells.

Nuclear dimensions in mammalian cells appear as a determining factor of chromatin organization and cellular radiosensitivity. Most radioresistant interphase cells have a nuclear volume (Vn) of 75 to 2700 micron 3, that could allow both the topological organization of chromatin as loops attached to the inner surface of the nuclear envelope and the unfolding of condensed chromatin within the topological constraints existing along the DNA molecule. In contrast, the radiosensitive small lymphocytes, with Vn values of 20 to 65 micron 3, seem to comprise significant amounts of highly condensed chromatin and dispose of an uncompleted topological organization of DNA, which may cause their incapacity to perform replication and transcription of DNA as well as the repair of radiation damage at a cell level. The indications are that radiosensitivity (1/D37) of animal cells, containing a similar quantity of DNA, should be directly proportional to 1/nuclear volume (1/Vn). However, DNA is unevenly distributed within the nuclear space, according to a partial ordering of interphase chromosomes; and it appears that radiosensitivity increases in zones of high DNA or chromatin density.

Animals↗

PubMedDate not supplied

[Structure of the macronucleus chromatin of the ciliate Bursaria truncatella. III. Chromatin organization in resting cysts and during excysting].

Structural organization of macronuclear chromatin of a ciliate Bursaria truncatella was studied electronmicroscopically by means of Miller's technique and negative staining of resting cysts and at excysting. In resting cysts practically all the macronuclear chromatin was shown to be organized into compact chromatin clumps 100-300 nm in size. At excysting a natural decompactization of the chromatin clumps occurred and radial loop-shaped chromatin fibres appeared around the clumps. Sometimes transcription units with a relatively small contour length of transcribed region and with high RNA-polymerase density were observed on the loops. The responsibility of such genes for the synthesis of proteins taking part in regulation of excystment and/or subsequent cell growth and differentiation is discussed.

Animals↗