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Biomedical subjects

G Russev

Publications and source records attributed to G Russev.

At least 37 records · Page 2Linked to original sources
PubMed1996

Immunofluorescent study after transient transfection of myoblast cells with the plasmid PSV40-beta-globin.

The level of transfection of myoblast C-2/C-12 cells with the plasmid PSV40-beta-globin, using the immunofluorescence assay, was investigated. Myoblasts, which are non-terminally differentiated cells, were chosen since it is known that terminally differentiated myotubes are very poorly transfected. The results showed that myoblast cells of the C-2/C-12 cell line were readily transfected within 24 h with the PSV40-beta-globin gene. The immunofluorescence assay showed the nucleoplasmic localization of the SV40 large T antigen, and indicated the possibility that some fraction of it might be localized in the cytoplasm.

Animals↗
PubMed1996

The influence of prostaglandin E1 on in vitro transcription of sperm chromatin from healthy males.

Run-on transcription experiments with sonicated chromatin from healthy individuals in the presence and absence of prostaglandin E1 showed different levels of transcription. Thus the level of incorporation of 14C-UTP into pre-mRNA proved to be about twice as high in the presence of postaglandin E1 as in the controls. The kinetic data of the in vitro transcription indicated that there were two peaks of incorporation of 14C-UTP into RNA both in the controls and in prostaglandin E1 treated probes but at different time intervals. The results show that prostaglandin E1 has a stimulatory effect on in vitro transcription of chromatin, isolated from normal patients.

Alprostadil↗
PubMed1995

Electron microscopic demonstration of transcription of ram sperm chromatin.

Transcription of ram sperm chromatin was examined by two electron microscopic techniques, namely spreading and thin sections. Labelling by Streptavidin-gold particles permits identification of transcription complexes, that have previously incorporated biotinylated uridine triphosphate. This supports previous electron microscopic data for randomly distributed transcription complexes and the presence of polymerase II molecules, documented by means of specific antibody using immunoelectron microscopy. Labelling of transcripts with biotin permits exact visualization of the transcription process, as well as identification of regions, where transcription occurs.

Adenosine Triphosphate↗
PubMed1995

Electron microscopic data for the presence of post-meiotic gene expression in isolated ram sperm chromatin.

A whole mount electron microscopic technique facilitated a direct visualization of transcripts in ram spermatozoa in run-on experiments. The localization of transcripts in sperm chromatin by spreading enabled identification of regions where transcription complexes, presumably pre-mRNA species, were seen related to chromatin. In another series of experiments the localization of polymerase II was demonstrated using a specific antibody against the conservative tail domain of the polymerase II molecule, followed by protein A-gold visualization on spread chromatin and on thin sections from mature spermatozoa. Incorporation of bio-UTP in transcripts was visualized by streptavidin-gold on spreading and on thin sections. The data suggest that transcription occurs at the periphery of the mature spermatozoa.

Animals↗
PubMed1995

Influence of freezing at different temperatures on the transcription activity of buffalo sperm chromatin.

The effect of freezing at different temperatures, -20 degrees C, -70 degrees C and -196 degrees C, on the transcription activity of buffalo sperm chromatin was investigated. The kinetic data of incorporation of 3H-UTP in pre-mRNA indicate that temperatures of -70 degrees C and -196 degrees C are the most favourable for preservation, since transcription activity gradually increases with time, reaching maximum values within 30 min. Freezing at -20 degrees C or preservation at 4 degrees C leads to a rapid decrease of transcriptional activity within 10 and 20 min, respectively. It is suggested that the optimal temperature for storage of buffalo spermatozoa is below -20 degrees C as judged by the transcription assay.

Animals↗
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↗
PubMed1993

Isolation and cloning of putative mouse DNA replication initiation sites: binding to nuclear protein factors.

By using an original two-step technique (trioxsalen crosslinking/immunoprecipitation) we were able to isolate in a single-stranded form a fraction of mouse DNA enriched in putative Replication Initiation Sequences (RIS). The isolated and purified single-strand fragments were made double-stranded in vitro and were cloned in pUC12 to prepare a confined RIS library. 30 randomly selected RIS inserts were subjected to gel mobility shift assay using nuclear extracts either from dividing, or from quiescent mouse cells. Twelve out of the 30 RIS fragments showed specific binding to proteins present in nuclear extract from dividing cells, while none were retarded by extracts from quiescent cells. RIS12, RIS18 and RIS30 were sequenced and it was found that they were A+T rich and contained different regulatory elements. By using a two step procedure (Heparin-sepharose chromatography/DNA affinity chromatography) we isolated the protein factor that specifically binds to RIS12. It appeared as a double band with apparent molecular masses of 63 and 65 kD.

Animals↗
PubMed1993

Effect of ionizing radiation and topoisomerase II inhibitors on DNA synthesis in mammalian cells.

We have used a novel, quantitative approach to study the effect of gamma-radiation and topoisomerase-II inhibitors on the initiation of DNA synthesis in eukaryotic cells. We found out that mild gamma-irradiation caused an almost immediate decrease in the rate of initiation of genomic DNA replication and stimulated DNA repair. This held true for two different cell lines. Ehrlich ascites tumor cells and Friend transformed erythroid cells, although the effect of gamma-radiation on Friend cells was more pronounced. At the same time, the synthesis of mitochondrial DNA was not affected by the irradiation. The effect of topoisomerase-II inhibitors on DNA initiation closely paralleled that of gamma irradiation, but did not stimulate repair. The fact that gamma-radiation and topoisomerase-II inhibitors, two types of agents that differ so profoundly, have practically the same effect on DNA synthesis speaks strongly in favour of the idea that eukaryotic cells have a general mechanism for coping with any disturbances in DNA integrity and chromatin structure. This mechanism is probably similar to the SOS-mechanism of prokaryotic cells and includes, as an early step, a slowdown of the initiation of replicative DNA synthesis.

Amsacrine↗
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↗