Metaphase Chromatin Research Articles

Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency.

TSG101 is a recently discovered tumor susceptibility gene whose functional inactivation in mouse fibroblasts results in cell transformation and the ability to form metastatic tumors in nude mice. Although restoration of TSG101 activity reverses tumorigenesis, neoplasia is irreversible in some cells, suggesting that permanent genetic alteration can occur during TSG101 inactivation. Here we describe studies that support this notion. We find that localization of TSG101 is cell cycle-dependent, occurring in the nucleus and Golgi complex during interphase, and in mitotic spindles and centrosomes during mitosis; cells made neoplastic by a deficiency in TSG101 expression show a series of mitosis-related abnormalities, including multiple microtubule organizing centers, aberrant mitotic spindles, abnormal distribution of metaphase chromatin, aneuploidy, and nuclear anomalies. Our findings suggest that TSG101 deficiency may lead to genome instability in addition to previously reported reversible neoplastic transformation.

Ligation-Mediated PCR for Chromatin-Structure Analysis of Interphase and Metaphase Chromatin

Chromatin structure is becoming increasingly recognized as important for a full understanding of gene function and cell memory. With regard to cell memory, which involves the transfer of chromatin-encoded epigenetic information from one cell generation to another, the detailed structure of metaphase chromatin is of crucial importance. In this paper we describe methods for the use of dimethyl sulfate, DNase I, and potassium permanganate forin vivofootprinting and chromatin analysis, with special emphasis on studies of metaphase cells. We review the use of ligation-mediated PCR for the analysis of chromatin, including the human phosphoglycerate kinase promoter, and also report initial studies of a matrix attachment region near the human β-interferon gene.

Assignment of rat thromboxane synthase gene (Tbxas) to chromosome 4q21-->q22 by fluorescence in situ hybridization.

Thromboxane A2 plays physiological and pathophysiological roles in many tissues. We isolated a cDNA for rat thromboxane synthase from the kidney and here show the location of rat thromboxane synthase gene (Tbxas) on rat chromosome 4q21-->q22 by fluorescence in situ hybridization to metaphase chromatin of rat lymphocytes.

Interphase-like chromatin configuration induced by cycloheximide in maturing pig oocytes: effects of protein phosphatase inhibitors.

Embryo cloning methods could greatly benefit from the manipulation of cell cycle in oocytes from large domestic mammals. The present study was undertaken to examine the effects of the protein synthesis inhibitor cycloheximide and the inhibitors of protein phosphatases 1 and 2A okadaic acid and calyculin A on maturing pig oocytes. Cycloheximide treatment (10 micrograms/ml) induced an interphase-like chromatin configuration (ICC) in maturing oocytes. Up to 69% of the oocytes exhibited ICC when treated with cycloheximide after 24 h of in vitro culture. ICC starts to appear after a 4 h exposure to cycloheximide and the ICC percentage reached its plateau after 12 h of cycloheximide treatment. ICC is fully reversible. The addition of okadaic acid (0.5 microM) inhibited the ICC in cycloheximide-treated maturing oocytes and allowed the completion of maturation in 55% of them. In oocytes with ICC, the immunocytochemistry for tubulin revealed the rearrangement of microtubule into an interphase meshwork and these oocytes lost their ability to induce tubulin assembly, as shown after short-time taxol treatment. The addition of okadaic acid prevented this microtubule rearrangement and preserved a certain level of tubulin assembly. Calyculin appeared to be more effective than okadaic acid in the prevention of ICC. It is concluded that de novo protein synthesis is necessary during a certain period of meiotic maturation for the maintenance of metaphase chromatin configuration in pig oocytes. This protein (or proteins) acts through the inhibition of endogenous protein phosphatases, probably protein phosphatase of 2A type.

Metaphase chromosome analysis by ligation-mediated PCR: heritable chromatin structure and a comparison of active and inactive X chromosomes.

We report that ligation-mediated PCR (LMPCR) can be used for high-resolution study of metaphase chromosomes, and we discuss the role of metaphase chromatin structure in the preservation of differentiated cell states. The X chromosome-linked human PGK1 (phosphoglycerate kinase 1) promoter region was investigated, and euchromatic active X chromosome (Xa) metaphase chromatin was compared with interphase Xa chromatin and to heterochromatic inactive X chromosome (Xi) metaphase and interphase chromatin. We find that (i) good-quality data at single-nucleotide resolution can be obtained by LMPCR analysis of dimethyl sulfate-treated intact metaphase cells; (ii) transcription factors present on the Xa promoter of interphase chromatin are not present on metaphase chromatin, establishing that the transcription complex on the PGK1 promoter must form de novo each cell generation; and (iii) the dimethyl sulfate reactivity pattern of Xa and Xi chromatin at metaphase is very similar to that of naked DNA. These results are discussed in the context of models for heritable chromatin structure and epigenetic mechanisms for cell memory, and they are also relevant to more general aspects of chromatin structure and differences between euchromatin and heterochromatin.

Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization

The objective of these experiments was to evaluate factors affecting in vitro fertilization of bovine oocytes matured in vitro, and their subsequent development to blastocysts. In Expts 1 and 2, sperm concentration, spermatozoa and oocyte incubation time, motility enhancers and semen source were manipulated. Fluorescent microscopy of microtubules and chromatin was used to observe sperm penetration rate, sperm aster formation and chromatin decondensation. Oocyte penetration rates were affected by sperm concentration but not by spermatozoa and oocyte incubation time. The effect of sperm concentration was due primarily to changes in polyspermy and not monospermy. Motility enhancers had no effect on any parameter measured. In Expt 3, oocytes were matured for 17, 22, 28 and 34 h before fertilization and evaluated for fertilization rates, morphology of cortical granules and exocytosis and blastocyst development. A domain free of cortical granules that was associated with the metaphase chromatin was not observed in mature bovine oocytes. As oocytes matured from 17 to 34 h, the distribution of cortical granules progressed from clustered to diffuse. Although monospermic fertilization rates were similar and cortical granule exocytosis occurred in all groups, polyspermy increased with maturation time. Development to blastocysts increased from 17 to 22 h of maturation but decreased thereafter with increasing maturation time. These results suggest that polyspermy can be reduced by adjusting sperm concentration and spermatozoa and oocyte incubation time with little effect on monospermic fertilization. Increased polyspermy with increased maturation time was not due to a lack of cortical granule exocytosis.

Diffuse kinetochores and holokinetic anaphase chromatin movement during mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC‐20

Mitosis in the hemipteran Agallia constricta (leafhopper) cell line AC-20 was examined by light microscopy of living and fixed cells. During early prometaphase the numerous small (0.30-3.0-microns) chromosomes appear as discrete units that lack a primary constriction. However, by late prometaphase the chromosomes are tightly packed at the spindle equator and are no longer clearly resolvable as individuals. When viewed from the side the metaphase chromatin appears as a 2-3-microns wide band that spans the width of the spindle; when viewed from the pole it appears as a fenestrated disk. The metaphase chromatin splits at anaphase into two sister chromatin plates, each of which exhibits holokinetic poleward movement, i.e., all parts of each plate move as a single unit with the same velocity. In many early-to-mild anaphase cells the separating sister plates are connected by chromatin-containing bridges that break as anaphase progresses. Ultrastructural analyses of serial thick and thin sections from cells fixed by conventional, OsO4/KFeCN, or high pressure rapid freezing methods, reveal that by metaphase all of the chromosomes are interconnected to form a large, irregularly shaped fenestrated disk of chromatin. Similar analyses reveal that adjacent chromatids remain interconnected throughout anaphase. Each disk of metaphase and anaphase chromatin contains numerous kinetochores recessed within its pole-facing surface. Kinetochores consist of a fine, faintly staining fibrillar material arranged along the chromatin surface as thin (0.1-0.3 micron dia.) rods varying considerably (0.15-2.3 microns) in length. From these observations we conclude that the polycentric metaphase chromatin of A. constricta, and its holokinetic behavior during anaphase, arises from the aggregation or cohesion of smaller prometaphase chromosomes, each of which contains a single, diffuse kinetochore.

Membrane-mediated changes in the structure of chromosomes

In the present paper the interaction of metaphase chromosomes and chromatin with model and natural lipid membranes was studied. It was shown that chromatin and chromosomes are able to form complexes with membranes in the presence of divalent cations. In such complexes, the typical structure of chromosomes is altered. The character of this alteration in chromosomal structure was investigated with the use of electron microscopy and chemical modification with dimethylsulphate (DMS). The latter is possible because, according to the presented data, the condensation of chromatin into chromosomes is associated with a decrease in accessibility of N-3 in adenine (the protection of the minor groove of DNA) to modifications, and with an increased methylation of N-1 in adenine (the disarrangement of the secondary structure of DNA). It was shown that the interaction of chromosomes with liposomes provides various levels of unfolding up to the appearance of chromatin-like structures. The secondary DNA structure of decondensed chromosomes coincides with the secondary structure of chromosomal but not chromatin DNA, whereas the extent of shielding of the minor groove of DNA in such decondensed structures typical for chromatin DNA. It is possible to suggest that the chromosomal decondensation in telophase of mitosis is initiated by the action of a membrane component of the developing nuclear envelope.

Immunocytochemical localization of cytoskeletal proteins and histone 2B in isolated membrane-depleted nuclei, metaphase chromatin, and whole Chinese hamster ovary cells.

Immunocytochemical techniques employing protein A-gold labeling were used to locate actin, tubulin, and histone 2B in thin sections of embedded, isolated membrane-depleted nuclei, metaphase chromosomes, and whole Chinese hamster ovary (CHO) cells. Actin and tubulin were detected in significant amounts in the cytoplasm and the nucleus of whole cells. The isolated membrane-depleted nuclei and metaphase chromosomes showed levels of actin and tubulin labeling either comparable to or sometimes lower than the levels of labeling in whole interphase cells. The results suggest that actin and tubulin are normal components of nuclei throughout the cell cycle. A mouse monoclonal antibody to histone 2B gave relatively weak labeling of nuclei and chromosomes in whole cells but intense labeling of isolated nucleoids and chromosomes. An increased concentration of antibody at the periphery of interphase nucleoids was noted.

Isolation of chromosome clusters from metaphase-arrested HeLa cells.

We have developed a simplified approach for the isolation of metaphase chromosomes from HeLa cells. In this method, all the chromosome from a cell remain together in a bundle which we call a "metaphase chromosome cluster". Cells are arrested to 90-95% in metaphase, collected by centrifugation, extracted with non-ionic detergent in a low ionic strength buffer at neutral pH, and homogenised to strip away the cytoskeleton. The chromosome clusters which are released can then be isolated in a crude state by pelleting or they can be purified away from nearly all the interphase nuclei and cytoplasmic debris by banding in a PercollTM density gradient. -- This procedure has the advantages that it is quick and easy, metaphase chromatin is recovered in high yield, and Ca++ is not needed to stabilise the chromosomes. Although the method does not yield individual chromosomes, it is nevertheless very useful for both structural and biochemical studies of mitotic chromatin. The chromosome clusters also make possible biochemical and structural studies of what holds the different chromosomes together. Such information could be useful in improving chromosome isolation procedures and for understanding suprachromosomal organisation of the nucleus.

Involvement of higher order chromatin structures in metaphase chromosome organization

Using electron microscopy we show that the metaphase chromatin fibers of Chinese hamster ovary cells form the same ionic strength-dependent higher order structures as the corresponding interphase chromatin fibers. We present evidence that such intact chromatin fibers are a prerequisite for the maintenance of the characteristic shape of metaphase chromosomes. The evidence is based on the finding that treatment of chromosomes with 0.5 M NaCI, a condition which is known to remove histone H1 and which destroys the higher order structure of chromatin fibers, also leads to a disintegration of the metaphase chromosome structure, whereas treatment with 0.3 M (or less) NaCI has no effect on the integrity of the chromosomes and their chromatin fibers. These data support a model in which the metaphase chromosome is maintained by a tight assembly of the 25–30 nm thick chromatin fibers containing all the histones.

Preparation of metaphase chromatin of [formula omitted] without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold Physarum polycephalum> which retains the in vivo property of RNA synthesis is described. Preparations from G 2-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg ++, deoxycholate and EGTA, a Ca ++-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg ++.

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 ⩾ mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.

Interphase and metaphase chromatin: Different stainability of DNA with acridine orange after treatment at low pH

The stainability of DNA in situ with acridine orange is different in interphase and mitotic cells. The difference is evident after pretreatment of cells with buffers at low pH (1.3–1.9). When acid treatment is followed by cell staining at neutral pH, the DNA of cells in mitosis, in comparison with DNA of interphase cells, exhibits lower green fluorescence and similar red fluorescence. Apparently partial extraction of acid-soluble constituents of chromatin at pH 1.3–1.9 increases the accessibility of DNA for AO intercalation and this increase is more extensive in interphase than in mitotic cells. When acid-treated cells are stained at pH 2.3–3.2, the DNA of mitotic cells in comparison with DNA of cells in interphase shows lower green fluorescence and markedly higher red fluorescence. It is presumed that staining at low pH potentiates acid denaturation of DNA in situ (and/or precludes the fast reassociation of the denatured DNA) and that the acid denaturation of DNA is more extensive in metaphase chromatin. Due to differences in the stainability of DNA in interphase vs mitotic cells, it is possible to discriminate between cells in G1, S, G2 and M phases, respectively. Consequently, the method may be used to automatically analyse the cell cycle including measurements of mitotic indices.

Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation.

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.

Synchronization of events in fused interphase-metaphase binucleate cells: progression of the telophase-like nucleus.

After the initial formation of nuclear envelopes around the metaphase chromosomes and the production of a telophase-like nucleus (TLN) 30 minutes after metaphase- interphase cell fusion, the chromatin of the TLN underwent structural and functional changesthat resembled the progression of a telophase nucleus in the normal cell cycle. To identify the metaphase chromatin, metaphases prelabeled with 3H-thymidine were fused with unlabeled interphase cells. Light microscopy of the TLN, up to 60 minutes after the initial formation, revealed a gradual loss of chromatid structure; by 60 minutes, the chromatin resembled that of interphase. Concomitantly, the number of recognizable TLN's fell, which was matched by an increase in the number of binucleate interphase-interphase cells containing only 1 radiolabeled nucleus. Electron microscopy revealed progressive formation of nucleolilike bodies and, 60 minutes after TLN formation, areas of decondensed chromatin. The ability of the TLN to incorporate 3H-uridine into its chromatin during its progression revealed a temporal pattern similar to that of the metaphase-G1 progression in the normal cell cycle. The progression of the TLN is another example of intracellular synchronization of cell cycle stages in virus-fused cell.

Fate of chromatin of interphase nuclei subjected to "phosphasing" in virus-fused cells.

The fate of chromatin of interphase nuclei (G1, S, or G2) of cells fused by means of Sendai virus with metaphase cells has been studied in a Chinese hamstercell line (DON). “Prephasing” of such interphase nuclei was differentiated from the metaphase chromosomes by 3H-thymidine labeling of their DNA before cell fusion. After prophasing reached its peak, Colcemid was removed from the culture to permit the binucleate cells to enter a G1 state. Interphase S or G1 chromatin became randomly distributed to daughter cells, when metaphase chromosomes underwent karyokinesis or subsequent cytokinesis, and persisted either as nuclear fragments or micronuclei. In a subsequent nuclear division, Sand G1 chromatin again were subject to prophasing and emerged again as “pulverized” chromatin, an indication of continuousasynchrony with the original metaphase nucleus. This contrasts with the behavior of G2 chromatin. The latter amalgamated with the metaphase chromatin to form a nucleus-like structure. In subsequent mitoses, most G2 chromatin was reorganized as intact metaphase chromosomes among those representative of the original metaphase cell. In this case, the G2 chromatin became synchronized with the original metaphase chromatin. Thus, providing the interphase nucleus is in G2, prophasing in a binucleate cell containing a metaphase set of chromosomes can be followed by events simulating metaphase as well. It is concluded that the metaphase cell contains factors capable of inducing many events of a normal mitosis.

F1-histone modification at metaphase in Chinese hamster cells

When the histones of metaphase and interphase Chiness hamster, HeLa S 3 and rat nephroma cells are compared by polyacrylamide gel elecrophoresis, a shift to slower mobility of metaphase F1-histone is observed. This change in mobility of the F1-histone results from enhanced phosphorylation since it is not observed after treatment of the extracted proteins with alkaline phosphatase. The phosphorylation is found in mitotic cells collected without use of a metaphase arrest agent. Further, the extent of phosphorylation of F1 is independent of the elapsed time of metaphase arrest with vinblastine. The phosphoryl-rich F1 subcomponents are dephosphorylated upon allowing the metaphase cell to enter the G 1 phase. The observed protein phosphorylation may be related to the unique physical state of metaphase chromatin. It has a pronounced influence on the chemical extraction of lysine-rich histone during isolation of metaphase chromosomes.

The surface and internal structure of metaphase chromatin.

Mammalian metaphase chromatin has been isolated by an ultrasonication technique and examined by both surface scanning and high-voltage electron microscopy (H.V.E.M.). By both techniques, rod-like structures 0.5 to 0.8 μ wide and varying in length from 3 to 5 μ were seen. Evidence is submitted that these represented parts of metaphase chromosomes. — By scanning microscopy the rods showed repeated patterns of wide and constricted areas. The constrictions were spaced 3,000 to 4,000 A apart and the entire surface of the rods was covered with smaller rounded projections. In addition, longitudinal grooves were occasionally seen. — H.V.E.M. revealed gross folded and unfolded fibres whose sizes correlated with the surface projections seen by scanning microscopy. In addition, microfibrils, 20–40 A in diameter were seen. The possibility that these fibrils represent the DNA co-helix is discussed.

Chemical differentiation with fluorescent alkylating agents in Vicia faba metaphase chromosomes

With the purpose of searching for chemical bases for the transfer of differentiation information through metaphase, efforts have been made cytochemically to identify chemical and physiochemical differences between different parts of metaphase chromatin in Vicia. Equipment has been developed for very high resolution quantitative spectrophotometric determinations on chromosomes and for fluorimetric work with low irradiation load on the object. A bifunctional highly fluorescent quinacrine mustard was shown to give chromosome breaks preferentially localized to heterochromatic chromosome regions (identifiable by cold treatment) in the M-chromosome. This and other fluorescent mustards of different types accumulated preferentially in the same regions, which then appear as strongly fluorescent bands. In these bands the fluorescence per unit DNA is higher than in the rest of the chromosome. Possible mechanisms for the fluorochrome-binding are discussed.