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L Vassilev

Publications and source records attributed to L Vassilev.

16 recordsLinked to original sources
PubMed1995

Sequence context of antisense RelA/NF-kappa B phosphorothioates determines specificity.

The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.

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↗
PubMed1990

An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells.

Studies on origins of DNA replication in mammalian cells have long been hampered by a lack of methods sensitive enough for the localization of such origins in chromosomal DNA. We have employed a new method for mapping origins, based on polymerase chain reaction amplification of nascent strand segments, to examine replication initiated in vivo near the c-myc gene in human cells. Nascent DNA, pulse-labeled in unsynchronized HeLa cells, was size fractionated and purified by immunoprecipitation with anti-bromodeoxyuridine antibodies. Lengths of the nascent strands that allow polymerase chain reaction amplification were determined by hybridization to probes homologous to amplified segments and used to calculate the position of the origin. We found that DNA replication through the c-myc gene initiates in a zone centered approximately 1.5 kilobases upstream of exon I. Replication proceeds bidirectionally from the origin, as indicated by comparison of hybridization patterns for three amplified segments. The initiation zone includes segments of the c-myc locus previously reported to drive autonomous replication of plasmids in human cells.

Cell Division↗
PubMed1989

Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments.

We describe a sensitive method for mapping replication initiation sites near regions of sequenced genomic DNA in vivo. It is based on selective amplification of sets of segments in purified nascent DNA strands and subsequent determination of the lengths of these strands required to include each member of the set. We demonstrate the ability of this method to accurately map a well-defined origin, that of replicating SV40 DNA. Pulse-labeled DNA from infected CV-1 cells was size-fractionated on an alkaline sucrose gradient and newly-synthesized strands purified by immunoprecipitation using anti-BrdU antibodies. Three pairs of synthetic oligonucleotide primers were used to amplify three SV40 segments, using the polymerase chain reaction (PCR), at known distances from the origin. Lengths of the nascent DNA strands that allow amplification were determined by hybridization to probes homologous to the amplified segments and used to calculate position of the origin. Experiments with a mix of SV40 and human HeLa cell DNA demonstrate the applicability of the method to mapping origins present at the level of single-copy genomic sequences in mammalian cells.

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↗
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↗
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↗
PubMed1980

Histone exchange in chromatin of hydroxyurea-blocked Ehrlich ascites tumour cells.

It is well established that DNA and histone synthesis are tightly coupled. Nevertheless, these two processes can be partially uncoupled by drugs specifically inhibiting protein or DNA synthesis, and also during n-butyrate-induced differentiation of Friend cells. The fate of the histones synthesized in the absence of DNA synthesis is unknown; they could be: (1) degraded without joining chromatin; (2) deposited on chromatin as extra histones; or (3) replace original chromatin histones. The only data concerning this problem are a recent report supporting the second possibility. We present evidence here in favour of the third possibility by showing that the histones synthesized in the absence of DNA synthesis enter chromatin and become organized in nucleosomes.

Animals↗
PubMed1980

Salt-induced structural changes in nucleosomes.

Ehrlich ascites tumor (EAT) nucleosomes treated with increasing NaCl concentrations were analyzed by sucrose density gradient centrifugation. Two events were found to take place in the course of the salt treatment: a) increasing amounts of nucleosomes dissociated into free DNA and protein in the interval 0.6M-1.5M NaCl, and b) the sedimentation coefficient of the nucleosomes decreased from 11S to 8S in the interval 0.6M - 1M NaCl. This decrease was not caused by loss of protein and was fully reversible upon slow and gradual lowering of the ionic strength. This shows that before dissociation of the protein core from DNA, nucleosomes undergo a structural transition. The electron microscopic observations revealed that it consisted in detachment of the ends of nucleosomal DNA from the protein core. It is suggested that an arginine-rich domain in the protein core exists, which holds more tightly the central part of the nucleosomal DNA, while its ends are relatively loosely bound to lysine-rish domains.

Animals↗