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Biomedical subjects

G Russev

Publications and source records attributed to G Russev.

At least 55 records · Page 3Linked to original sources
PubMed1988

U4 and U6 small nuclear ribonucleoprotein particles. 1. Fractionation and characterization of snRNPs containing U4 and U6 RNAs.

The isolation of U4 and U6 small nuclear ribonucleoprotein particles (snRNP) was undertaken, since there has been no reliable method for their fractionation established. The procedure subjects a nuclear extract from HeLa cells to several types of ion exchange chromatography at moderate ionic strength, electrophoresis on agarose gels, transfer of the particles on DEAE cellulose paper, and elution with ammonium chloride. The purified U4 and U6 snRNPs contain U4 and U6 RNAs, respectively, and a set of six polypeptides, present in U4 RNPs and seven polypeptides in U6 snRNP particles. Five major proteins with molecular masses of 70, 64, 47, 40.5 and 24 kD are common to both assemblies. In addition U4 RNPs contain one polypeptide of 25.1 kD which is unique for this class of particles, while U6 RNPs possess two polypeptides with molecular masses of 31 and 18 kD which are not present in U1, U2 or U4 snRNPs. The U4 and U6 RNP particles thus fractionated retain their antigenicity as judged by their reaction with auto-antibodies from patients with lupus erythematosus.

Chromatography, Affinity↗
PubMed1988

Isolation of U5 snRNP particles.

A method for the isolation of intact U5 small nuclear RNP particles from HeLa cells has been developed. The procedure includes nuclear extraction of the particles at moderate ionic strength, fractionation in agarose gels, electrophoretic transfer on DEAE cellulose paper and elution with ammonium chloride. The purified U5 snRNP particles contain U5 RNA and a set of eleven proteins. They retain their antigenicity as judged by their reaction with autoantibodies from patients with connective tissue diseases.

Cell Fractionation↗
PubMed1988

U5 snRNP particles bind pre-messenger RNA in vitro.

The ability of purified U5 small nuclear RNP particles (U5 snRNPs) to bind in vitro pre-mRNA of the human beta-globin gene was investigated. The transcript which contained sequences, corresponding to the second intron and fragments of the second and third exons of the gene was bound to U5 snRNP particles in the presence of antibodies specific against U5 snRNPs. The in vitro splicing of pre-mRNA was inhibited in the presence of autoantibodies against U5 snRNP particles. The results are discussed in terms of the possible role of U5 RNP particles in pre-mRNA splicing.

Antibodies↗
PubMed1986

Distribution of the short dispersed repetitive sequences B1 and B2 in the mouse genome.

The organization of the short dispersed repetitive sequences B1 and B2 in the mouse genome was investigated by hybridization of randomly selected genomic clones with isolated and labelled in vitro B1 and B2. Cloning and restriction mapping experiments indicated that these two DNA sequences were not entirely independently distributed along mouse DNA, but approximately half of them formed heterologous pairs separated by stretches of apparently random DNA.

Animals↗
PubMed1985

Identification of the short dispersed repetitive DNA sequences isolated from the zones of initiation of DNA synthesis in human cells as Alu-elements.

DNA of Xeroderma pigmentosum cells was crosslinked in vivo with trioxsalen and long wave length ultraviolet light and the cells were cultured in the presence of labelled thymidine for one hour. The nascent DNA chains synthesized during this period and containing the DNA replication origins were isolated from the high molecular weight chromosomal DNA by an alkaline sucrose density gradient centrifugation. They were 5-10-fold enriched in short dispersed repetitive sequences identified by dot-blot hybridization to BLUR 8 plasmid as members of the human Alu-family.

DNA↗
PubMed1985

Determination of certain physical and chemical characteristics of the activating serum factor from preparturient women and women with habitual abortions.

Follow-up investigation of the blood sera from preparturient women and women with habitual abortions showed the presence of a factor which has an activating effect on smooth muscle preparations because it causes the release of prostaglandins. Gel-chromatographic counter flow separation and microelectrophoresis of the blood sera have shown that the isolated serum factor is a water soluble glycopeptide with a molecular weight of about 2000.

Abortion, Habitual↗
PubMed1984

Random distribution of mammalian replication origins in matrix and total nuclear DNA.

Nuclear matrices from mouse and rat tumour cells were isolated and characterized by their microscopic appearance, protein profiles and DNA content. They presented well-defined structures containing 15-20% of the nuclear protein and 1-3% of the nuclear DNA. Matrix DNAs were immobilized on nitrocellulose filters and hybridized to nick-translation 32P-labelled homologous DNA fragments containing the corresponding replication origins. As control total nuclear DNAs were also immobilized on filters and hybridized to origin-containing DNAs. The origin-containing DNAs hybridized to the same extent to both matrix and total DNAs, which showed that they contained the same proportion of origin sequences. In an alternative series of experiments, plasmids containing either rat or mouse replication origins were immobilized on filters and were hybridized with in vitro 32P-labelled matrix and total nuclear DNAs. Here again both matrix and total nuclear DNAs hybridized to the same extent with the origin-carrying plasmids, which showed that neither rat nor mouse matrix DNAs were enriched in DNA replication origin sequences.

Animals↗
PubMed1984

Isolation of short interspersed repetitive DNA sequences present in the regions of initiation of mammalian DNA replication.

Nascent DNA chains containing the putative replication origins were isolated from cells of human embryonic lung fibroblasts, Hela, Ehrlich ascites tumour and Guerin ascites tumour as described earlier [ Russev , G., and Vassilev , L. (1982) J. Mol. Biol. 161, 77-87]. It was demonstrated that the synthesis of these nascent chains correlated with the ability of cells to initiate semiconservative DNA replication. Reassociation and electrophoretic analysis showed that the nascent chains from all four cell lines contained middle repetitive DNA in the form of short interspersed sequences. Mouse repetitive sequences were isolated and hybridized to Escherichia coli, chicken, calf and rat DNA and to homologous hnRNA. The kinetics of hybridization indicated that the repetitive sequences found in the vicinity of the replication origins were order-specific and were not heavily transcribed. Reassociation experiments, in which homologous DNA isolated from nuclei digested with micrococcal nuclease to different extents was used as a driver, showed that these repetitive sequences were organized into nucleosomes like the bulk of the chromatin.

Animals↗
PubMed1984

Hydroxyurea treatment does not prevent initiation of DNA synthesis in Ehrlich ascites tumour cells and leads to the accumulation of short DNA fragments containing the replication origins.

The ability of EAT cells to initiate DNA synthesis in the presence of high doses of hydroxyurea was examined using the recently developed method for crosslinking DNA in vivo. Since crosslinking blocks elongation but has little effect on initiation (Russev and Vassilev (1982) J. Mol. Biol. 161, 77-87), this approach permits a separate study of the two stages of the DNA replication. We found out that hydroxyurea did not greatly affect the initiation of DNA replication but strongly inhibited the elongation of the already initiated new DNA chains. This resulted in the formation of short fragments enriched in sequences synthesized at and around the sites where DNA initiation began. These fragments were not ligated to the high molecular weight chromosomal DNA and could be released under denaturing conditions in single-stranded form. The reassociation and electrophoretic analysis showed that they contained about 200 nucleotides long interspersed DNA sequences repeated approx. 10(4) times per haploid genome, that probably served as replication origins.

Animals↗
PubMed1983

Differential binding of nonhistone chromosomal proteins to the putative mouse origin of replication.

Ehrlich ascites tumour (EAT) cells were treated with trioxsalen and ultraviolet light to crosslink DNA in vivo. After the treatment initiation of DNA replication can still occur but elongation is blocked by the crosslinks and this leads to the formation of short DNA fragments containing the origin of replication that can be isolated in double-stranded form after S1 nuclease cutting of the crosslinked DNA (Russev, G. and Vassilev, L. (1982) J. Mol. Biol. 161, 77-87). To assess the affinity of these DNA fragments toward different chromosomal proteins, chromatin was fractionated by SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose filters and allowed to interact with in vivo labelled [32P]DNA. The autoradiography of the filters showed that the DNA fraction synthesized between crosslinks and containing the putative mouse origin of replication bound preferentially to several nonhistone proteins, the most strongly binding ones having molecular weights of 64, 68, 72 and 150 kDa.

Animals↗
PubMed1983

Labelling pattern of the nonhistone chromosomal proteins in quiescent and dividing Ehrlich ascites tumour cells.

Upon pulse-labelling with [14C]protein hydrolizate both in dividing and quiescent Ehrlich ascites tumour (EAT) cells the nonhistone chromosomal (NHC) proteins had uniform specific radioactivity with few exceptions: Both in quiescent and dividing EAT cells a polypeptide with molecular weight of about 200 kdalton had specific radioactivity 2-3 times lower than that of the most of the NHC proteins. In the actively proliferating cells a group of proteins with molecular weights between 45 and 65 kdalton had 2-3 times higher specific radioactivity than most of the NHC proteins. In quiescent cells the specific radioactivity of a group of proteins with molecular weights in the range 18-25 kdalton was 3-4 times higher than that of the rest of the NHC proteins.

Animals↗
PubMed1983

Comparative study of the tightly bound nonhistone chromosomal proteins of rat liver and Ehrlich ascites tumour cells.

Nuclei from rat liver and Ehrlich ascites tumour (EAT) cells were fractionated into nuclear matrix and soluble chromatin. The tightly bound nonhistone chromosomal (NHC) proteins of the corresponding chromatin fractions were isolated and analyzed by means of SDS-polyacrylamide gel electrophoresis. The comparison of the electrophoretic profiles revealed that these tightly bound proteins consisted of few major fractions common to both rat liver and EAT cells.

Animals↗
PubMed1982

Organization of the newly replicated chromatin in the vicinity of the replication fork.

Ehrlich ascites tumour cells were pulse-labelled with [3H]thymidine for 1 min or were treated with cycloheximide and labelled with [3H]thymidine for 45 min. The kinetics of digestion with micrococcal nuclease of both pulse-labelled and cycloheximide chromatins showed that they exhibited increased susceptibility towards the enzyme. At the same time their release from the nucleus was retarded and this was interpreted to mean that, unlike the bulk of chromatin, they were tightly bound to a fixed nuclear structure. When subjected to an equilibrium metrizamide-triethanolamine density gradient centrifugation both pulse-labelled and cycloheximide chromatins banded at higher density than control chromatin, which was an indication of their higher protein to DNA ratio. After a mild trypsinization, eliminating H1 and the nonhistone proteins, the pulse-labelled chromatin sedimented to the same density as control chromatin, and the cycloheximide chromatin sedimented to a density which was intermediate between those of control chromatin and free DNA. This result showed that the newly replicated chromatin had the same, and the cycloheximide chromatin half the amount of core histones present in control chromatin.

Animals↗
PubMed1982

Assembly of new histones into nucleosomes and their distribution in replicating chromatin.

We studied the assembly of new histones into nucleosomes and their distribution in replicating chromatin in growing P815 mouse cells. New histones and new DNA were density-labeled with 13C, 15N, 2H-substituted amino acids together with [3H]arginine or with 5-iododeoxyuridine and [3H]thymidine, respectively, for 1 hr (approximately 20% of S phase). Mono- di-, tri-, tetra- and larger oligonucleosomes were isolated by sucrose gradient centrifugation of micrococcal nuclease-digested chromatin, and their density distribution was analyzed, without fixation, in metrizamide/triethanolamine density gradients [Russev, G. and Tsanev, R. (1976) Nucleic Acids Res. 3, 697-707] in which mono- and oligonucleosomes containing dense amino acids or 5-iododeoxyuridine separate from the corresponding normal nucleosomes. Under these conditions, approximately 74% of the new histones are found in nucleosomes on newly replicated DNA, and the remainder are on unreplicated DNA. The majority of new histones form entirely new nucleosomes; a minor fraction may form hybrid nucleosomes that also contain preexisting histones. New nucleosomes are distributed to both new daughter DNA molecules with approximately equal probability, and our evidence suggests, but does not prove, that they are distributed in a random manner along new DNA.

Animals↗
PubMed1981

Formation of hybrid nucleosomes cantaining new and old histones.

5 mM hydroxyurea (HU) inhibits DNA synthesis in mouse P815 cells by 94-97% in less than 1 hr. Nevertheless, histone synthesis continues and newly-synthesised histones are incorporated into non-replicating chromatin at a rate of about 20% of that in control exponentially-growing cells. To study the organization of these histones in chromatin P815 cells were treated with 5 mM HU in medium containing dense (15N, 13C, 2H) - substituted amino acids. After inhibition of DNA synthesis, newly-synthesised histones were labelled with (3H)-arginine. The cells were harvested 90 min later, and mono- and oligonucleosomes were prepared and analysed on metrizamide-triethanolamine (MA-TEA density gradients. Analysis of the distribution of 3H-labelled histones in these gradients shows that they are incorporated into hybrid mononucleosomes containing both new and old histones. It is also shown that these hybrid nucleosomes are not randomly distributed, but show a certain tendency to be clustered in certain chromatin regions.

Animals↗