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Biomedical subjects

G Russev

Publications and source records attributed to G Russev.

At least 19 recordsLinked to original sources
PubMed1994

Isolation and purification of U7 snRNP particles.

1. A procedure has been developed for the isolation of U7 small nuclear ribo-protein particles involved in the processing of histone pre-mRNAs. They were fractionated from the rest of the snRNPs through a series of gel filtration and affinity chromatography steps. 2. The isolated assembly of U7 snRNP particles appeared to be intact and pure as judged by the gel electrophoresis of their RNA. No detectable RNA species were monitored other than U7 RNA.

Chromatography, Gel↗
PubMed1994

Characterization of the protein moiety of U7 small nuclear RNP particles.

U7 snRNP particles contain a set of seven proteins with molecular weights in the range of 13.5 to 50 kD. One protein with a molecular weight of 18 kD carried the antigenic determinant responsible for the interaction of U7 snRNPs with autoantibodies from patients with connective tissue diseases. It is suggested that U7 RNA is associated with proteins representing a ribonucleoprotein particle analogous to U1, U2, U4, U5 and U6 snRNPs.

Autoantibodies↗
PubMed1994

Isolation and purification of U7 and snRNP particles.

A procedure was developed for the isolation of U7 small nuclear ribonucleoprotein particles involved in the processing of histone pre-mRNAs. U7 snRNP particles were fractionated from the rest of the snRNPs by means of a series of gel filtration and affinity chromatography steps. The isolated assembly of U7 snRNP particles appeared to be intact and pure as judged by gel electrophoresis of their RNA. No detectable RNA species were monitored other than U7 RNA.

Cell Fractionation↗
PubMed1994

The effect of three cryoprotective diluents on the in vitro transcription of ram sperm cells.

The effect of three different solvents was investigated in run-on experiments, using purified ram sperm nuclei. The best cryoprotective diluent proved to be Nagase-Niwa, which had the most positive effect on transcription. The kinetic data of incorporation of 32P-ATP in pre-mRNA indicated that there was a stepwise increase in the level of transcription during the first 20 min of incubation, being lowest in the control and highest in the presence of the diluent Nagase-Niwa.

Animals↗
PubMed1992

Repair of transcriptionally active and inactive genes during S and G2 phases of the cell cycle.

To study the effect of ultraviolet irradiation on S and G2 phases of the cell cycle, BB88 mouse cells synchronized by a double thymidine block were exposed to ultraviolet light, and rates of DNA synthesis and mitotic indexes were determined at regular intervals. It was found that with increasing ultraviolet dose, semiconservative DNA synthesis decreased and the sharp mitotic wave observed in the unirradiated cells gradually degenerated. To study repair, semiconservative DNA replication was inhibited with hydroxyurea at different time intervals after releasing cells from the block and the DNA synthesized as a result of repair of the ultraviolet damage was labeled with 5'-bromodeoxyuridine (BrdU). The newly repaired DNA was separated from bulk DNA by immunoprecipitation with monoclonal anti-BrdU antibody, labeled with 32P and hybridized to nine different gene and oncogene probes dot-blotted in excess on nylon membranes to determine their abundance in the repaired DNA. The results showed that: (a) the most actively repaired segment was a 211-bp sequence adjacent to the promotor region of the beta-actin gene; (b) all transcriptionally active genes were repaired at similar and constant rates throughout S and G2 phases; (c) the nontranscribed genes were repaired at much lower rates in early S phase, but later in S phase and especially in G2 phase, their repair rates increased and approached those of the transcribed genes.

Animals↗
PubMed1991

3' processing of histone H4 precursor mRNA requires the presence of a small nuclear RNP particle.

1. Incubation of in vitro synthesized mouse histone H4 mRNA precursors in nuclear extracts of mouse 3T6 fibroblasts, rat L6-5 myoblasts and myotubes yields processed mRNA species. A processing activity was identified in all three kinds of extracts that cleaves the precursor transcripts, generating mRNA species with mature 3' termini. 2. The processing activity is present both in proliferating (mouse 3T6 fibroblast, rat L6-5 myoblast) and terminally differentiated (rat L6 myotube) cells. 3. The efficiency of the endonucleolytic cleavage reactions was higher in a homologous system, i.e. in the presence of nuclear extracts from mouse 3T6 fibroblasts, than in a heterologous system, i.e. in the presence of rat myoblast or myotube extracts. 4. The in vitro processing activity is specifically inhibited by anti-Sm antibodies, which suggests the requirement of an snRNP particle for H4 pre-mRNA maturation.

Animals↗
PubMed1991

U7 snRNP particles bind H4 pre-mRNA in vitro.

1. U7 snRNP particles were bound in vitro to H4 pre-mRNA using immunoassay experiments. 2. The processing of in vitro H4 pre-mRNAs is an SmRNP-dependent reaction. 3. U7 RNA was isolated as a unique species from the complex H4-pre-mRNA-snRNPs, which indicates its involvement in the processing of histone pre-mRNAs.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

DNA replication and poly(ADP-ribosyl)ation of chromatin.

The role of poly(ADP-ribosyl)ation in chromatin replication and the activity of poly(ADP-ribose) synthetase in the newly synthesized and old chromatin was studied. It was found that 3-aminobenzamide, which is an inhibitor of poly(ADP-ribose) synthetase, had no effect on the initiation of DNA synthesis and only a moderate effect on DNA chain elongation. However, poly(ADP-ribose) synthetase activity in the newly replicated chromatin was two to three times higher than that of the unreplicated chromatin.

Animals↗
PubMed1988

Kinetics of replicon initiation during S phase of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were synchronized by a thymidine-hydroxyurea block. At different times after release from the block, cells were treated with trioxsalen and long-wavelength ultraviolet light to crosslink DNA in vivo and were labelled with [3H]thymidine for 30 min. This technique permits labelling of only the short nescent DNA fragments initiated between crosslinks. The amount of radioactivity incorporated in these fragments during the labelling period reflects the number of replicon initiation events and allows us to follow the replicon initiation pattern after removing the inhibitor. It was shown that the rate of initiation was high at the beginning of S phase and then steadily decreased.

Animals↗
PubMed1988

U4 and U6 small nuclear ribonucleoprotein particles. 2. Evidence for their involvement in pre-mRNA splicing.

The in vitro splicing of pre-mRNA of the human beta-globin gene in the presence of HeLa cell nuclear extract was investigated. Splicing was inhibited by auto-antibodies against U4 and U6 snRNP particles. No intermediates or products of the splicing reaction were evident in the presence of antibodies against U4 and U6 snRNPs which suggests their involvement in pre-mRNA splicing.

Antibodies↗
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↗