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Biomedical subjects

J Yaneva

Publications and source records attributed to J Yaneva.

16 recordsLinked to original sources
PubMed1998

Linker histone binding to superhelical DNA: electrophoretic and filter binding studies.

It has been known for years that linker histones bind preferentially to supercoiled DNA. This preference has been demonstrated by a number of different techniques including deoxyribonucleoprotein electrophoresis, sedimentation, and filter binding under non-competitive conditions. In an attempt to further study this issue, we used one- and two-dimensional electrophoretic gels and filter binding under competitive conditions, with all DNA forms of interest being simultaneously present in the incubation mixture. Comparison between results obtained by the two methods showed that whereas the preference for superhelical molecules was clearly seen in the electrophoretic gels, the filter binding assay failed to reveal this preference. These results reveal limitations to the filter binding technique, which must be borne in mind in studies involving superhelical DNA molecules.

Animals↗
PubMed1998

Proteins that specifically recognize cisplatin-damaged DNA: a clue to anticancer activity of cisplatin.

Cisplatin, but not its trans geometric isomer, is a potent anticancer drug whose biological activity is a consequence of the formation of covalent adducts between the platinum compound and certain bases in DNA. Two classes of proteins have recently been identified that bind preferentially to damaged sites: proteins that specifically recognize those sites as a first step in their repair, and those that bind to such sites by virtue of structural similarity between the modified DNA and their own natural binding sites. Both classes of proteins may be involved, perhaps in opposing ways, in the cytotoxic effect of the drug.

Antineoplastic Agents↗
PubMed1997

The major chromatin protein histone H1 binds preferentially to cis-platinum-damaged DNA.

Both cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) and trans-diamminedichloroplatinum(II) form covalent adducts with DNA. However, only the cis isomer is a potent anticancer agent. It has been postulated that the selective action of cis-DDP occurs through specific binding of nuclear proteins to cis-DDP-damaged DNA sites and that binding blocks DNA repair. We find that a very abundant nuclear protein, the linker histone H1, binds much more strongly to cis-platinated DNA than to trans-platinated or unmodified DNA. In competition experiments, H1 is shown to bind much more strongly than HMG1, which had been previously considered a major candidate for such binding in vivo.

Animals↗
PubMed1996

Linker histones inhibit T4 and Escherichia coli DNA ligases.

Based on some preliminary observations that linker histones strongly inhibit the activity of prokaryotic DNA ligases, we studied the effect of these histones on the ligation of short restriction DNA fragments by either T4 or E. coli DNA ligases. The inhibitory effect was strong, but it appeared only after two molecules of H1 bound to a approximately 200 bp-long DNA fragment. A similar pattern of inhibition (but at much higher concentration) was observed with the isolated globular domain of histone H5. That the inhibition was specific to the linker histones became clear when other basic proteins, such as the core histone octamer or cytochrome C, were tested. They did not inhibit the ligases but rather significantly stimulated them. The other major linker DNA-binding protein in chromatin, the non-histone protein HMG1, showed no significant effect on the ligase activity.

Animals↗
PubMed1996

Effect of mechanical pressure on c-fos and on the mitotic activity of epidermal cells.

A dosed mechanical pressure of 12.5 kg/cm2 applied for 1 min on depilated mouse skin did not cause cellular death or visible alterations of the cellular ultrastructures. However, it had a strong effect on the mitotic cycle of the epidermal cells-stimulating the cells to enter the mitotic cycle and temporarily blocking the G1-->S transition. This effect was strictly limited to the pressed area of the skin. The proto-oncogene c-fos was induced within the first 2 min following application of the pressure. The level of c-fos mRNA showed two peaks during the next 24 h. The first slight peak was preceded by a rapid increase in the cAMP level in the pressed skin, the second-by a fall in the cAMP concentration. A model is suggested to explain the observed effects by reversible functional damage of the cellular membrane affecting the enzymes maintaining the steady state level of cAMP.

Animals↗
PubMed1995

High-affinity binding sites for histone H1 in plasmid DNA.

The interaction of histone H1 isolated from chicken erythrocytes with restriction fragments from plasmids pBR322 and pUC19 was studied by gel electrophoresis. Certain restriction fragments exhibited unusually high affinity for the histone, forming high molecular mass complexes at protein to DNA ratios at which the other fragments did not show evidence for binding. The highly preferred fragments are intrinsically curved, as judged by their electrophoretic mobility in polyacrylamide gels, by computer modeling, and by imaging with scanning force microscopy. However, control experiments with either curved portions of the same fragments or highly curved kinetoplast DNA fragments showed that the presence of curvature alone was not sufficient for preferential binding. By using various restriction fragments centered around the highly preferred sequence, it was found that the high-affinity binding required in addition the presence of specific sequences on both sides of the region of curvature. Thus, both curvature and the presence of specific sites seem to be required to generate high affinity.

Animals↗
PubMed1994

A spot test for protein detection and semiquantitative estimation in small samples.

Modern biological experiments, conducted on mini- and micro-scales require methods for protein detection in small samples. The method proposed here is based on visual inspection of protein dots immobilized on nitrocellulose filters and stained with the common protein dye amido black. It allows detection and semiquantitative determination of protein concentrations in a wide range.

Amido Black↗
PubMed1992

An indirect-labeling procedure for the study of specific interactions of proteins with DNA.

A semiquantitative and reproducible indirect-labeling procedure for the study of specific protein/DNA interactions using nitrocellulose-filter-immobilized proteins and linear or superhelical DNA molecules is reported. Proteins were immobilized on nitrocellulose filters either by direct dotting or by electrotransfer from polyacrylamide electrophoretic gels. After incubation with the respective DNA (linear restriction fragments or closed circular recombinant DNA plasmids) the paper strips were washed, and specifically bound DNA was denatured by alkali and detected by hybridization with 32P-nick-translated DNA. The quantitation of the reaction was performed by scanning of the autoradiograms of the radioactive spots and determination of the area under the respective peaks. The intensity of the radioactive spots was proportional to the amount of protein present in the dot. The sensitivity of the assay depends primarily on the affinity of the respective DNA to the protein and in the case of mouse liver histone H1AB/mouse alpha-globin gene approximately 50 ng of protein per dot was enough for determination.

Animals↗
PubMed1992

Histone H1 interacts specifically with certain regions of the mouse alpha-globin gene.

We used fragments of a cloned mouse alpha-globin gene to determine if histone H1 interacts selectively with defined regions of a eukaryotic gene. The use of intact plasmids instead of isolated fragments permitted study of relevant sequences in their superhelical form. Several independent experimental approaches (filter binding, precipitation, binding to protein immobilized on nitrocellulose membranes, and agarose gel electrophoresis of the protein-DNA complexes) were used and the histone-DNA interaction was investigated under both noncompetitive and competitive conditions. Binding to subclones encompassing the 5' end of the gene and the first half of the coding sequence is preferred over binding to other subclones. The expression of the sequence-specific selectivity depends on the ionic strength of the binding reaction; the selectivity is mainly expressed under conditions of non-cooperative binding of the histone to DNA. No correlation is observed between AT content and relative affinity of binding to H1. Evidently, other features of DNA structure are involved in the specific H1 binding.

Animals↗
PubMed1991

DNA sequence specific interactions of histone H1.

Histone H1 is know to play a role in the formation and maintenance of higher-order chromatin structure. It has been recently suggested that the linker histone might be also involved in the regulation of the activity of individual genes. If H1 is a regulatory factor in eukaryotic gene transcription, it should possess specificity of binding to defined DNA sequences. This review is an attempt to summarize and discuss the existing literature data on DNA sequence-specific interactions of histone H1.

Animals↗
PubMed1991

Histone H1-DNA interactions and their relation to chromatin structure and function.

The belief that histone H1 interacts primarily with DNA in chromatin and much less with the protein component has led to numerous studies of artificial H1-DNA complexes. This review summarizes and discusses the data on different aspects of the interaction between the linker histone and naked DNA, including cooperativity of binding, preference for supercoiled DNA, selectivity with respect to base composition and nucleotide sequence, and effect of H1 binding on the conformation of the underlying DNA. The nature of the interaction, the structure of the complexes, and the role histone H1 exerts in chromatin are also discussed.

Chromatin↗
PubMed1990

Interaction of histones H1 and H1(0) with superhelical and linear DNA.

By using direct competition experiments, the binding of histone H1AB (a mixture of H1A and H1B) and H1(0) to superhelical and linear DNA forms was studied. Mouse liver H1 isohistones and plasmid p alpha GD containing part of the 5' flanking and part of the coding sequence of the mouse alpha-globin gene in pUC18 were used as partners in the binding reaction. The competition experiments were performed by direct mixing of the histone with labelled supercoiled DNA (at 125 mM NaCl and at a histone/DNA ratio of 1.0) and addition to the mixture of increasing amounts of cold competitor DNA, either supercoiled or linear. The radioactivity of the complex formed was determined by filter binding. The results show that both histones H1 and H1(0) posses a strong binding preference for supercoiled DNA forms. Thus, histone H1(0) resembles the regular somatic set of histone H1 and not the other differentiation-specific histone H5 studied thus far.

Animals↗
PubMed1989

Some features of DNA fragments associated in vivo with the nuclear lamina.

Ehrlich Ascites Tumour cells were irradiated with UV-light to crosslink DNA to proteins in vivo. The DNA fragments associated with the nuclear lamina were purified and characterized. The results of the Cot analysis and the hybridization experiments suggest that the DNA fragments attached to the nuclear lamina although containing the entire complexity of genomic DNA are enriched in some highly repeated sequences.

Animals↗
PubMed1987

Segregation of nucleosomes in replicated mouse alpha-globin gene.

DNA of mouse erythroleukemia cells grown in vitro was labeled with bromodeoxyuridine during cycloheximide-inhibited protein synthesis. Isolated nuclei were digested with micrococcal nuclease to obtain monosomes and monosomal dsDNA. The protection of the heavy and of the light strands of the newly replicated DNA was studied by dot hybridization with the coding and with its complementary noncoding strand of the alpha-globin gene. The results show that both sides of the replication fork contain protected sequences of the gene, thus supporting a bilateral (dispersive) mode of nucleosome segregation during DNA replication.

Animals↗
PubMedDate not supplied

The electrophoretic separation of curved cisplatin-modified DNA fragments on polyacrylamide gels is dependent on the voltage gradient.

Polyacrylamide gel electrophoresis has been widely used to study DNA fragments containing sequence-dependent curvature. The anomalous electrophoretic behavior of curved DNA fragments on such gels allows their separation from straight fragments of the same length. Here we demonstrate that polyacrylamide gels can be successfully used to resolve DNA fragments modified at a single site by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) from their unmodified counterparts. However, the resolution strongly depends on the voltage gradient, being completely lost when it drops below a certain threshold level. The parameters of the electric field do not affect separation of 'normal' DNA fragments of comparable length.

Base Sequence↗