Lalio Djondjurov

Nuclear matrix and transcriptional activity of the mouse α-globin gene

Abstract The association of the mouse α-globin gene with the nuclear matrix was studied when the gene was permanently repressed in Ehrlich ascites cells, potentially active in uninduced Friend cells or actively transcribed in induced Friend cells. Matrix-associated DNA was obtained by two methods, differing in the order of treatment of the nuclei with high salt and DNase I. By using a cloned α-globin probe, no enrichment in α-globin sequences was found in the matrix-associated DNA after DNase I digestion of high-salt treated nuclei from Ehrlich ascites and uninduced Friend cells. In induced Friend cells, a high enrichment (up to 20 times) of α-globin sequences was detected in the DNA left with the nuclear matrix structures. The size of the DNA fragments obtained by this procedure indicated a random attack and did not correspond to a progressive top-to-bottom cleavage model. No enrichment in α-globin sequences was found in induced Friend cells if nuclear matrices were obtained by DNase I digestion of the nuclei before the treatment with high salt. Our data suggest that the enrichment in actively transcribed genes of matrices from nuclei treated with high salt does not reflect a localization of these genes close to the attachment sites of the chromatin loops but rather their artefactual association with some high salt-insoluble proteins of the transcriptional complexes.

Is the nuclear matrix the site of DNA replication in eukaryotic cells

Four types of experiment were carried out to test the recently proposed model of matrix-bound replication in eukaryotic cells. In experiments with pulse-labelling we found preferential association of newly replicated DNA with the matrix only when the procedure for isolation includes first high-salt treatment of isolated nuclei and then digestion with nucleases, or when prior to digestion the nuclei have been stored for a prolonged time. In both cases, however, evidence was found that this preferential association is due to a secondary, artifactual binding of the newly replicated chromatin region to the matrix elements. Pulse-chase experiments and experiments with continuous labelling were carried out to answer the question whether during replication the DNA is reeled through the replication complexes, i.e., whether newly replicated DNA is temporarily or permanently associated with the matrix. The results showed that at that time the matrix DNA does not move from its site of attachment. Since, according to the model of matrix-bound replication, the forks are assumed to be firmly anchored to high-salt resistant proteinaceous matrix structures, the chromatin fragments isolated with endonuclease not recognizing newly replicated DNA and purified by sucrose gradient centrifugation should be free of replication intermediates. The electronmicroscopic analysis of such fragments revealed the existence of intact replication micro-bubbles. Moreover, the fragments with replication configurations appeared as smooth chromatin fibres not attached to elements characteristic for the matrix. All these experiments suggest that the nuclear skeleton is not a native site of DNA replication in eukaryotic cells.

Cell cycle dependent turnover of non-histone proteins in chromatin

Abstract Chinese hamster fibroblasts were labelled with 14C-tryptophan and 3H-thymidine during the logarithmic growth phase. The decay of 14C and 3H radioactivity in isolated chromatin and in the fraction of non-histone proteins was monitored during the subsequent stationary phase and after resumption of cell proliferation. The results show the presence of non-histone proteins which are metabolized only during the mitotic cycle. A subfraction of this group is completely eliminated during the mitotic cycle, while another subfraction is conserved like histones.

Distribution of tryptophan-containing proteins and of newly synthesized RNA in metaphase chromosomes: An autoradiographic study

Abstract Chinese hamster fibroblasts were labelled with 3 H-tryptophan (for 15.5 h), with 3 H-uridine (for 2 h) and with 3 H-thymidine (for 15.5 h) in vitro. The distribution of the label was studied by autoradiography of isolated chromosomes. While 3 H-thymidine-labelled chromosomes showed the well known uniform distribution of the label, in chromosomes labelled with 3 H-tryptophan the label was unevenly distributed along the chromosomes. Quantitative measurements of the grain density over different segments of two easily identified chromosomes showed that each chromosome had a characteristic labelling pattern. 3 H-uridine was incorporated in the same regions where 3 H-tryptophan was localized. Control experiments showed that the observed labelling pattern was not due to non-specific adsorption of cytoplasmic ribonucleoproteins.

A method for recovery of proteins from sodium dodecyl sulfate-polyacrylamide gels

Abstract A procedure is described for recovery of proteins after separation in analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A high percentage of recovery is achieved (60–80%) for the range from 5–50 μg protein loaded, allowing the quantitation recovered proteins with standard Lowry method. The procedure is designed for the calculation of specific radioactivities of labeled proteins in order to measure their half-lives. For the purpose of illustration a description is given of the recovery of labeled HeLa nuclear matrix proteins.

Macromolecular and ultrastructural organization of the mitotic chromosome scaffold

Using electron microscopy (EM), we have examined three structural domains of the mitotic chromosome scaffold of mouse erythroleukemia (MEL) Friend cells with different morphologic organization: centromeric, intermediate, and telomeric. The intermediate, most extensive, domain exhibited a specific fibrogranular structure representing tightly packed granular bodies with diameters between 20 and 60 nm. The chromosome scaffold contained three main components: proteins (81%), RNA (12%), and DNA (7%). The residual DNA extracted from the scaffold represented short fragments, 300 bp on average, belonging to the class of tandemly arranged repetitive DNA. In situ hybridization experiments confirmed its typical centromeric location. Scaffold RNA represented three fractions: a major RNA fraction with an electrophoretic mobility corresponding to that of 5S RNA and two minor fractions with electrophoretic mobilities somewhat lower than that of 18S RNA. Scaffold RNA was localized mainly in the centromeric region. We show that the newly synthesized protein component of the chromosome scaffolds migrates slowly to the chromosomes, reaching a maximum specific radioactivity 12 h from the onset of the chase period.

Isolation of chromatin fragments released by apoptotic cells

A two-step procedure for isolation of chromatin fragments released from apoptotic cells is described. The first step includes extraction of the chromatin fragments by a hypotonic treatment of isolated nuclei. The second step provides stabilization of the extracted chromatin fragments and their collection by a high-speed centrifugation. The procedure ensures not only a complete extraction and collection of the chromatin fragments but also intactness of their components. A number of control experiments confirmed this statement: they showed that the percentage of recovered fragmented DNA does not exceed 10% from the total DNA of apoptotic cells and that it is specific in both nature and chromosomal localization; the experiments reveal, moreover, that the protein components of the fragments including the well known histone and non-histone species possess also ability to support in in vitro conditions their specific phosphorylation. Developed for recovery of chromatin fragments from mouse erythroleukemia cells, spontaneously entering apoptosis, the procedure is practically applicable for all eukaryotic cell systems sharing programmable death.

Increased proteolysis in chromatin of terminally differentiated and quiescent cells

Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [3H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference--increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases--supported the latter alternative.

Metabolic behaviour of nonhistone chromosomal proteins during differentiation (spherulation) of Physarum polycephalum

Abstract 1. 1. Spherulation of Physarum microplasmodia is accompanied by a transient and moderate intranuclear accumulation of nonhistone proteins during the first 12 hr after induction. 2. 2. At least part of these proteins have been synthesized before induction but migrate to the nucleus after the transfer of plasmodia to non-nutrient medium. 3. 3. While in the cytoplasm at the final stage of sperulation most of the proteins have been replaced by new molecules, the majority of the nonhistone proteins synthesized before induction were found preserveclin the spherules.

Matrix-associated DNA from maize is enriched in repetitive sequences.

In order to elucidate some features of the topological organization of DNA within the plant nucleus, DNA fragments involved in the attachment of the DNA loops to the nuclear matrix in maize were studied. The matrix-associated DNA from dry embryo and meristematic cells after extensive digestion with DNase I and high salt treatment was about 2% of the total DNA, sized within the range of 50 and 250 bp. This DNA was found to be enriched in repetitive DNA sequences, both for nuclei from dry embryo and meristematic cells. The loop size of the DNA in cells of Zea mays appeared to be between 5 and 25 kbp.