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Biomedical subjects

R Tsanev

Publications and source records attributed to R Tsanev.

At least 37 records · Page 2Linked to original sources
PubMed1987

Differential DNase I sensitivity of the two complementary nucleosomal DNA strands in cycloheximide-treated Ehrlich ascites tumor cells.

The accessibility of the two complementary DNA strands in newly replicated chromatin of Ehrlich ascites tumor (EAT) cells grown under conditions of cycloheximide-inhibited protein synthesis was studied by analysis of the DNase I digestion of isolated nuclei. Bulk DNA was labeled with 14C-thymidine and the newly synthesized strands - with bromodeoxyuridine and 3H-thymidine. The DNase I digests were fractionated in two successive CsCl density gradient centrifugations to obtain a dense fraction containing 15-20% newly replicated DNA. Analysis of the distribution of 14C-labeled parental DNA fragments complementary to the 3H-nascent strand has shown that the 14C-labeled fragments prevail in the region of 30-50 nucleotides. Simulation experiments using the rate constants for DNase I attack show that this result may be explained by an enhanced accessibility at the nucleosomal 5'-end region of the parental strands, where the H2a-H2b dimer interacts with DNA. This asymmetry seems to be induced by interactions in the chromatin.

Animals↗
PubMed1986

Activated murine alpha-globin gene is not preferentially associated with the nuclear matrix.

The association of the murine alpha-globin gene with the nuclear matrix was studied in three different states of the gene: inactive (EAT cells), potentially active (MEL cells) and active (induced MEL cells). When "native" nuclei were digested with DNase I it was found that the nuclear matrix was not enriched in alpha-globin DNA sequences in all three different types of cells. A nuclease-hypersensitive site in the 5'-flanking region of the alpha-globin gene was detected in the induced MEL-cells.

Animals↗
PubMed1985

Presence of nucleosomal repeat in the transcribed alpha globin gene of induced murine erythroleukemia cells.

The sensitivity of the mouse alpha-globin gene to micrococcal nuclease and its nucleosomal repeat were studied in three different functional states of the gene: inactive in EAT cells, potentially active in uninduced MEL cells and active in induced MEL cells. The results show that: 1. The nuclease sensitivity of the gene differs in the three different functional states. 2. Both the coding and the 5'-flanking regions of the induced actively transcribed gene show a typical nucleosomal repeat pattern. 3. Hypersensitive sites for micrococcal nuclease and for an endogenous nuclease appear upstream of the gene after induction of differentiation.

Animals↗
PubMed1985

Differences in the mode of iodination of H2a variants in chromatin.

Modification of H2a variants with radioactive iodine was used to study under different ionic conditions the accessibility of their tyrosine residues in chromatin, in monosomes and when free in solution. The modification of tyrosine 57 in the hydrophobic part of H2a was found responsible for the appearance of new fractions with a reduced electrophoretic mobility in the presence of Trition X 100, detected only by autoradiography (radioactive "ghosts"). At low ionic strength a very small number of molecules were iodinated in chromatin, the modification affecting only their hydrophobic region. At moderate ionic strength the tyrosine residues near the N-terminal region of the molecule were predominantly modified. In chromatin the accessibility of the tyrosine residues of H2a1 was much greater than that of H2a2, a difference not observed with free histones.

Animals↗
PubMed1984

Nuclear matrix and transcriptional activity of the mouse alpha-globin gene.

The association of the mouse alpha-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 alpha-globin probe, no enrichment in alpha-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 alpha-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 alpha-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.

Animals↗
PubMed1984

Biochemical and ultrastructural study of the sperm chromatin from Mytilus galloprovincialis.

Protein composition and ultrastructure of the mature spermatozoa of the mussel Mytilus galloprovincialis were studied upon gradual decondensation of the nuclei with increasing NaCl concentration. Three types of protein were found, associated with the sperm DNA: (1) the sperm-specific proteins S1, S2 and S3 (80% of the acid-soluble proteins); (2) the four core histones (20%); (3) three non-histone proteins tightly bound to DNA (about 4 micrograms protein per 100 micrograms DNA). The sperm-specific protein S3 was the first to dissociate at about 0.5 M NaCl and electron micrographs of spread nuclei indicated its participation in the final compaction of the nucleus. Hypotonically treated sperm nuclei revealed the presence of 21-25 nm large granules irregularly scattered along some of the DNA fibers. These granules correspond to the 'superbeads' of histone-containing chromatins. The tightly bound non-histone proteins were represented by a triplet in the range 60-80 kD. They formed 30-60 nm large annular bodies holding DNA fibers and resisting high salt-detergent treatment.

Animals↗
PubMed1984

Distribution and metabolic behaviour of "tightly bound" acid-soluble non-histone chromosomal proteins in developing rat brain cells.

"Tightly bound" acid-soluble non-histone chromosomal proteins of rat brain were studied, and limited but detectable tissue specificity of their pattern was demonstrated. This class of proteins showed specific distribution in rat brain cells at different stages of development. The most prominent differences were observed between non-differentiated and terminally differentiated cells. In non-differentiated rat brain cells the acid-soluble non-histone protein fraction contained both metabolically labile and metabolically stable proteins, while in fully developed cells the main portion of acid-soluble proteins showed metabolic stability.

Aging↗
PubMed1984

Histone H1o in developing rat brain cells.

Histone H1o was found both in neuronal and oligodendrocyte rat-brain nuclei fractionated by sucrose-gradient isopycnic centrifugation. This histone was absent during the early stages of development when the brain cells were still proliferating, but it appeared in significant amounts in the terminally differentiated cells.

Aging↗
PubMed1983

Dynamics of H1(0) content in rat liver after partial hepatectomy.

The changes in the lysine-rich histone subfraction H1(0) have been quantitatively studied in rat liver during the regeneration period after partial hepatectomy. A gradual decrease in this protein was found early after operation with a minimal value around the time of maximal mitotic activity. The reduction in the H1(0) content paralleled well the increasing number of cells in the cell cycle.

Animals↗
PubMed1983

Intranuclear localization of DNA polymerases alpha and beta in regenerating rat liver.

1. Nuclei isolated from regenerating rat liver were digested with micrococcal nuclease and fractionated on glycerol gradients into soluble chromatin fragments and chromatin associated with the nuclear skeleton. 2. Distributions of DNA polymerases alpha and beta in these fractions were different. While beta polymerase followed closely the distribution of the chromatin fragments, alpha polymerase associated preferentially with the skeleton-chromatin complex. 3. At least 20% of total alpha polymerase in the nuclei was shown to be bound to the skeleton. In nuclei extracted with isotonic sucrose buffer containing 50 or 100 mM Tris-Cl the portion of the skeleton associated enzyme was increased to 40-50%. 4. These data show that the skeleton bound alpha polymerase was preferentially retained in the nuclei during salt extraction. 5. Contrary to the replicational DNA polymerase alpha, DNA polymerase beta did not show any affinity to the skeleton.

Animals↗
PubMed1983

Trout sperm chromatin. I. Biochemical and immunological study of the protein composition.

Compact sperm chromatin was obtained from mature trout sperm nuclei resistant to sonication and detergent treatments. 0.5 to 2 M NaCl caused a gradual decondensation of this chromatin and the dependence of the percentage of dissociated proteins on the salt concentration indicated cooperativity of the dissociation process. Urea alone was insufficient to decondense the nuclei. The only proteins dissociated from the sperm nuclei by NaCl alone or combined with urea were protamines. Besides protamines, tightly bound nonprotamine proteins resisting high salt-urea extraction were detected in the sperm nucleus. Part of them could be solubilized by 1% sodium dodecyl sulphate (SDS) and displayed the characteristics of the core histones: they were soluble in 0.25 N H2SO4, their electrophoretic mobilities were similar to those of trout liver core histones, and they shared common antigenic determinants with the latter. The rest of the tightly bound proteins resisted 1% SDS treatment and could be obtained after an extensive digestion of DNA with DNase I. These were nonhistone proteins similar in mobility to the protein triplet characteristic of the lamina-pore complex and an additional high molecular weight protein.

Animals↗
PubMed1983

Trout sperm chromatin. II. Ultrastructural aspects after salt dissociation of proteins.

The ultrastructural organization of the trout sperm nucleus was studied in ultrathin sections and spread preparations after partial decondensation of the nucleus with increasing NaCl concentrations. The obtained results suggest that the organization of the trout sperm chromatin is much more complex than a pure nucleoprotamine. Three types of complexes were observed. The first one results from the association of DNA with protamines. This complex appears as a fibrous network when partially decondensed nuclei are digested with DNase I indicating that at least a part of DNA remains protected by protamines and favours models accepting a colinear alignment of the latter on the DNA molecules. The second type of structures represent the DNA-protamine fibers compacted into dense clumps which appear as separate compaction units seen upon partial decondensation of the sperm nucleus. A third type are complexes of the ring-shaped granular bodies tightly associated with DNA and resisting high salt-urea and detergent treatment.

Animals↗