Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine

1992 ◽  
Vol 98 (1) ◽  
pp. 19-32 ◽  
Author(s):  
L. M. Neri ◽  
G. Mazzotti ◽  
S. Capitani ◽  
N. M. Maraldi ◽  
C. Cinti ◽  
...  
Keyword(s):  
Nuclear Matrix ◽  
Matrix Bound

scholarly journals Properties of chicken erythrocyte histone deacetylase associated with the nuclear matrix

1996 ◽  
Vol 314 (2) ◽  
pp. 631-637 ◽  
Author(s):  
Wei LI ◽  
Hou Yu CHEN ◽  
James R. DAVIE
Keyword(s):  
Molecular Mass ◽  
Histone Deacetylase ◽  
Nuclear Matrix ◽  
Active Form ◽  
Striking Difference ◽  
Ionic Strength Dependence ◽  
Strength Dependence ◽  
Solubilized Enzyme ◽  
Matrix Bound

Histone H2B is deacetylated more rapidly than H3 and H4 in chicken immature erythrocytes. Histone deacetylase from chicken immature erythrocytes was partially purified, and the histone specificities of the multiple histone deacetylase forms were determined. Ion-exchange (Q-Sepharose) and gel-exclusion (Superdex 200) chromatography of extracts from erythrocyte nuclei showed two forms (HD1 and HD2) of histone deacetylase. HD1, with a molecular mass of about 55 kDa, preferred free H3–H4 relative to H2A–H2B, while HD2, with a molecular mass of approx. 220 kDa, had a slight preference for H3–H4. HD1 and HD2 differed in pH- and ionic-strength-dependence. HD2 dissociated into HD1 when treated with 1.6 M NaCl or when applied to a Q-Sepharose column. The enzymic properties of nuclear-matrix-bound histone deacetylase showed a striking difference from that of HD1 and HD2, particularly in its strong preference for H2A–H2B. Treatment of the nuclear matrix with 1.6 M NaCl and 1% 2-mercaptoethanol solubilized histone deacetylase, which chromatographed as 400 and 220 kDa forms on a Superdex 200 column. The solubilized enzyme retained its histone preference for H2A-H2B. Chromatography of the nuclear-matrix-derived enzyme on Q-Sepharose yielded one peak of enzyme activity with chromatographic properties and histone specificities similar to those of HD1. These results provide support for the active form of the enzyme in situ being a high-molecular-mass complex associated with proteins that are components of the nuclear matrix. Substrate preference of the enzyme is governed by the proteins associated with the histone deacetylase.

Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum

1993 ◽  
Vol 105 (4) ◽  
pp. 1121-1130 ◽  
Author(s):  
S. Lang ◽  
T. Decristoforo ◽  
W. Waitz ◽  
P. Loidl
Keyword(s):  
Cell Cycle ◽  
Nuclear Matrix ◽  
Physarum Polycephalum ◽  
Nuclear Periphery ◽  
S Phase ◽  
Cycle Phase ◽  
Electron Microscopic ◽  
Nuclear Lamins ◽  
Synchronous Cell ◽  
Matrix Bound

We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually resembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycle-dependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues. In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.

In situ preparation of the nuclear matrix of Physarum polycephalum: ultrastructural and biochemical analysis of different matrix isolation procedures

1988 ◽  
Vol 90 (4) ◽  
pp. 621-628 ◽  
Author(s):  
W. Waitz ◽  
P. Loidl
Keyword(s):  
Nuclear Matrix ◽  
Physarum Polycephalum ◽  
Matrix Isolation ◽  
Protein Composition ◽  
Biochemical Properties ◽  
Agarose Beads ◽  
In Situ Preparation ◽  
The Matrix ◽  
Important Approach

A novel method for in situ preparation of nuclear matrix from whole plasmodia of Physarum polycephalum without isolation of nuclei is presented. Plasmodia are encapsulated in agarose beads and after solubilization of the cytoplasm the nuclear matrix is prepared. With this quick and easy technique nuclear matrix can be reproducibly prepared with perfect recovery. We compared the ultrastructural and biochemical properties of the matrix after three different matrix isolation procedures: preparation with high salt, ammonium sulphate and lithium diiodosalicylic acid. The results show that the ultrastructure and protein composition of the three types of matrix are very similar or even identical. We conclude that many of the conflicting results on nuclear matrix in the literature are due to perturbations of nuclear integrity during the isolation of nuclei. For this reason the new in situ method is an important approach in the standardization of nuclear matrix isolation.

A comprehensive study on the isolation and characterization of the HeLa S3 nuclear matrix

1991 ◽  
Vol 98 (3) ◽  
pp. 281-291
Author(s):  
P. Belgrader ◽  
A.J. Siegel ◽  
R. Berezney
Keyword(s):  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Structural Integrity ◽  
Rnase A ◽  
Matrix Proteins ◽  
Intermediate Filament Proteins ◽  
Isolation And Characterization ◽  
Hela S3

Different agents have been employed to extract the histones and other soluble components from isolated HeLa S3 nuclei during nuclear matrix isolation. We report that 0.2M (NH4)2SO4 is a milder extracting agent than NaCl and LIS (lithium 3,5-diiodosalicylate), on the basis of the apparent preservation of the elaborate fibrogranular network and the residual nucleolus that resemble the in situ structures in whole cells and nuclei, minimal aggregation, and sufficient solubilization of DNA and histones. The importance of intermolecular disulfide bonds, RNA and 37 degrees C stabilization on the structural integrity of the nuclear matrix was examined in detail using sulfydryl alkylating, reducing and oxidizing agents, and RNase A. The data suggest that any disulfides formed during the isolation are not essential for maintaining the structural integrity of the in vitro matrix. However, structural integrity of the matrix is dependent upon RNA and to some degree on disulfides that presumably existed in situ. Sodium tetrathionate and 37 degrees C stabilization of isolated nuclei resulted in nuclear matrices containing an approximately twofold greater amount of protein, RNA and DNA than control preparations. The 37 degrees C incubation, unlike the sodium tetrathionate stabilization, does not appear to induce intermolecular disulfide bond formation. Neither stabilizations resulted in significant differences of the major matrix polypeptide pattern on two-dimensional (2-D) gels stained with Coomassie Blue as compared to that of unstabilized matrix. The major nuclear matrix proteins, other than the lamins, did not react to the Pruss murine monoclonal antibody (IFA) that recognizes all known intermediate filament proteins, suggesting that the internal matrix proteins are not related to the lamins in intermediate filament-like quality.

Studies on Protein Subunits. V. Specific Interaction between Matrix–Bound Subunits of Aldolase and Soluble Aldolase Subunits

1973 ◽  
Vol 51 (9) ◽  
pp. 1240-1247 ◽  
Author(s):  
W. W.-C. Chan
Keyword(s):  
Specific Interaction ◽  
Saturation Level ◽  
Guanidinium Chloride ◽  
Protein Subunits ◽  
The Matrix ◽  
Tetrameric Structure ◽  
Matrix Bound ◽  
Small Aliquot ◽  
Kinetics Of

A derivative containing matrix-bound (MB) subunits of aldolase was prepared by attaching native tetrameric aldolase to Sepharose to form MB-aldolase followed by dissociation with 6 M guanidinium chloride and renaturation. The interaction between the MB-subunit aldolase derivative so prepared and subunits added in solution was studied. Nascent subunits of aldolase were generated in situ by diluting a small aliquot of guanidinium chloride-denatured aldolase into a much larger volume of buffer containing a suspension of MB-subunit aldolase. This treatment caused a significant increase in the amount of bound activity. The ability of MB-subunit aldolase to pick up activity from solution is highly specific. After repeated treatments with nascent sub-units, the matrix-bound activity reached a saturation level close to four times the activity of MB-subunit aldolase. The product of this treatment is very similar in properties to the original MB-tetrameric aldolase but different from MB-subunit aldolase. These observations suggest that MB-subunit aldolase can associate with nascent subunits generated in solution to form MB-tetrameric aldolase. The results in this paper support the conclusion from the kinetics of renaturation (Chan et al. J. Biol. Chem. 248, 2778 (1973)) that aldolase monomers have the same activity whether they exist singly or as part of a tetrameric structure.

scholarly journals Generation of an internal matrix in mature avian erythrocyte nuclei during reactivation in cytoplasts.

1983 ◽  
Vol 96 (6) ◽  
pp. 1815-1819 ◽  
Author(s):  
R E LaFond ◽  
H Woodcock ◽  
C L Woodcock ◽  
E R Kundahl ◽  
J J Lucas
Keyword(s):  
Nuclear Matrix ◽  
Rna Synthesis ◽  
New Method ◽  
Host Cytoplasm ◽  
L Cell ◽  
Novel Approach ◽  
Avian Erythrocyte

When fused with mouse L-cell cytoplasts, chick erythrocyte nuclei enlarge, take up proteins from the host cytoplasm, and recommence RNA synthesis. We found that during this transition the erythrocyte nuclei gain an internal nuclear matrix, thus providing a novel approach to questions concerning the nature of the salt-resistant intranuclear skeleton. A new method for preparation and examination of the nuclear matrix in situ is also described.

Progress in ultrastructural and molecular analysis of the nuclear matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 508-509
Author(s):  
Edward G. Fey
Keyword(s):  
Electron Microscopy ◽  
Nuclear Matrix ◽  
Structure And Function ◽  
Matrix Proteins ◽  
Rna Transcription ◽  
Nuclear Matrix Proteins ◽  
The Past ◽  
And Function

In the past few years, considerable advances have been made regarding the structure and function of the nuclear matrix. In the first half of this presentation, the field of nuclear matrix research will be summarized. Emphasis will be placed on those studies where molecular interactions are demonstrated in situ utilizing high resolution light and/or electron microscopy. Studies demonstrating the role of the nuclear matrix in DNA synthesis and replication, RNA transcription and processing, and the binding of matrix attachment regions to specific nuclear matrix proteins will be summarized.

Ki-67 detects a nuclear matrix-associated proliferation-related antigen. II. Localization in mitotic cells and association with chromosomes

1989 ◽  
Vol 92 (4) ◽  
pp. 531-540
Author(s):  
R. Verheijen ◽  
H.J. Kuijpers ◽  
R. van Driel ◽  
J.L. Beck ◽  
J.H. van Dierendonck ◽  
...  
Keyword(s):  
Nuclear Matrix ◽  
Fluorescence Signal ◽  
Ki 67 ◽  
Daughter Cells ◽  
Interphase Cells ◽  
Late Telophase ◽  
The Individual ◽  
Reticulate Structure ◽  
Mitotic Cells

In interphase cells the proliferation-associated antigen recognized by monoclonal antibody Ki-67 is almost exclusively located in the nucleoli. When cells at several stages of mitosis were examined for the localization of the Ki-67 antigen, a striking redistribution could be observed. During prophase the distinct nucleolar Ki-67 fluorescence changed to a bright irregular meshwork throughout the nucleoplasm. At metaphase the antigen appeared to be distributed in a reticulate structure surrounding the condensed chromosomes, while at late telophase a punctated staining of the entire nucleoplasm was observed, which preceded the typical nucleolar localization pattern in each of the two daughter cells. Immunolabelling with Ki-67 of metaphase chromosome spreads revealed a circumferential staining of the individual chromosomes. The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells. When mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold. Quantification of the Ki-67 fluorescence signal using flow cytometry revealed the highest staining intensities in mitotic cells. Furthermore, it was shown that nutritionally deprived cells became negative for Ki-67.

Sign in / Sign up

Export Citation Format

Share Document