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Biomedical subjects

I G Pashev

Publications and source records attributed to I G Pashev.

At least 19 recordsLinked to original sources
PubMed2001

In vivo acetylation of HMG1 protein enhances its binding affinity to distorted DNA structures.

The postsynthetic acetylation of HMG1 protein has been known for more than 20 years, but the effect of this modification on the properties of the protein has not been studied so far. Acetylated HMG1 was isolated from cells grown in the presence of sodium n-butyrate and identified as a monoacetylated protein, modified at lysine 2. Acetylated and parental forms of HMG1 were compared relative to their binding affinity to distorted DNA structures. By using mobility shift assay to determine the dissociation constants, we show that acetylation enhanced the ability of HMG1 to recognize UV light- or cisplatin-damaged DNA and four-way junctions. Since the modified lysine lies adjacent to the HMG1 DNA-binding domain, the results obtained were attributed to acetylation-induced conformational change in HMG1. The potential role of acetylation in modulating the interactions of HMG1 with both damaged DNA and other proteins is discussed.

Acetylation↗
PubMed1998

Preferential binding of high mobility group 1 protein to UV-damaged DNA. Role of the COOH-terminal domain.

Binding of chromosomal high mobility group 1 protein (HMG1) to UV-damaged DNA has been studied with oligonucleotides containing a single dipyrimidine site for formation of UV photolesions. Irradiation of an oligonucleotide with unique TT dinucleotide resulted in generation of cyclobutane pyrimidine dimer with no evidence for induction of (6-4) photoproducts, whereas the analysis of irradiated TC-containing oligonucleotide detected (6-4) photoproducts but not cyclobutane pyrimidine dimers. Mobility shift assays have revealed that HMG1 protein binds preferentially to irradiated TT and TC oligonucleotides. Photoreversal of cyclobutane pyrimidine dimers with DNA photolyase and hydrolysis of the (6-4) photoproducts with hot alkali substantially reduced but did not eliminate binding of HMG1. The protein, therefore, appears to bind the two main types of UV damages in DNA, but some other photolesion(s) contributes to the preferential binding of HMG1 to irradiated DNA. By quantifying gel shift assays and considering the efficiencies of lesion formation, we determined dissociation constants of 1.2 +/- 0.5 and 4.0 +/- 1.5 microM for irradiated TT and TC oligonucleotides, respectively, and 70 +/- 20 microM for the control non-irradiated probes. Tryptic removal of the acidic COOH-terminal domain of HMG1 significantly affected binding of the protein to both irradiated and intact oligonucleotides. The potential role of HMG1 in recognition of the UV lesions in DNA is discussed.

Animals↗
PubMed1996

Histones associated with non-nucleosomal rat ribosomal genes are acetylated while those bound to nucleosome-organized gene copies are not.

Acetylation of histones bound to rat rRNA genes has been studied relative to their organization in chromatin, either as canonical nucleosomes, containing the inactive copies, or as anucleosomal nonrepeating structures, corresponding to the transcribed genes (Conconi, A., Widmer, R. M., Koller, T., and Sogo, J. M. (1989) Cell 57, 753-761). Nuclei from butyrate-treated rat tumor cells were irradiated with a UV laser to cross-link proteins to DNA, and the purified covalent complexes were immunofractionated by an antibody that specifically recognized the acetylated histones. Upon probing with sequences coding for mature rat 28 S RNA, DNA of the antibody-bound complexes was 5-20-fold enriched relative to the total rat DNA. Since the laser cross-links histones to DNA in both active and inactive genes, one cannot distinguish which one of them, or both, are bound to acetylated histones. Alternatively, purified mononucleosomes were immunofractionated, but DNA from the antibody-bound monosomes was not enriched in coding rDNA. Taken together, these results suggest that nucleosome-organized rRNA genes are bound to nonmodified histones and that the acetylated histones are associated with the active, anucleosomal gene copies.

Acetylation↗
PubMed1992

Binding of histones to Xenopus laevis ribosomal genes with different levels of expression.

The association of ribosomal RNA genes with histones as a function of their expression has been studied in Xenopus laevis erythrocytes, where the genes are silent, and tadpoles at stage 40, where these genes are actively transcribed. Isolated nuclei were either treated with formaldehyde or irradiated with an ultraviolet laser to cross-link proteins to DNA. The covalently linked protein-DNA complexes were purified by centrifugation through CsCl and immunoprecipitated with antibodies against H1, H2A and H4. DNA from the precipitated complexes was analysed for the presence of ribosomal DNA sequences by hybridization to specific probes. The actively transcribed ribosomal genes from X. laevis embryos are associated with H1, H2A and H4 as are the non-transcribed genes in the erythrocytes.

Animals↗
PubMed1991

Crosslinking proteins to nucleic acids by ultraviolet laser irradiation.

Ultraviolet (UV) irradiation can initiate complex formation between proteins and DNA or RNA and so can be used to study such interactions. However, crosslink formation by standard UV light sources can take up to several hours. More recently, a beam of monochromatic UV light from a laser has been used to initiate crosslinking in nano- and picosecond time intervals. As noted in an earlier TIBS article 'the advantages of short pulse times and high-energy fluxes should make this a valuable technique in the future'. In this review we characterize laser-induced crosslinking and explore the applications of this method.

Adenosine Triphosphatases↗
PubMed1990

The enhancers and promoters of the Xenopus laevis ribosomal spacer are associated with histones upon active transcription of the ribosomal genes.

The presence of histones on the enhancer-promoter region of the X.laevis ribosomal spacer has been studied in embryos at stage 40, where the ribosomal genes are actively transcribed. Isolated tadpole nuclei were either fixed with formaldehyde or irradiated with UV laser to crosslink histones to DNA. The purified protein-DNA complexes were immunoprecipitated with antibodies to the histones H1, H2A and H4 and the DNA fragments carrying the respective histones were analyzed for the presence of spacer enhancer-promoter sequences by hybridization to specific DNA probe. The two independent crosslinking procedures revealed the presence of these DNA sequences in the precipitated DNA. The quantitative analysis of the UV laser-crosslinked complexes showed that histones H2A and H4 were associated with enhancer-promoter DNA in amounts similar to those found for bulk DNA, whilst the content of H1 was reduced.

Animals↗
PubMed1990

The chromatin fiber: structure and conformational transitions as revealed by optical anisotropy studies.

Structure and conformational transitions of the chromatin fiber as revealed by optical anisotropy studies are reviewed. The data in the literature do not allow a definite interpretation; in fact some of them are contradictory. The major findings are reported here and an attempt is made to analyse them with respect to the internal dynamics and the various models suggested for the organization of the chromatin fiber.

Cations↗
PubMed1989

Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.

UV laser irradiation has been used to covalently crosslink histones to DNA in nuclei, chromatin and core particles and the presence of the different histone species in the covalently linked material was detected immunochemically. When nuclei were irradiated and then trypsinized to cleave the N- and C- terminal histone tails, no histones have been found covalently linked to DNA. This finding shows that UV laser-induced crosslinking of histones to DNA is accomplished via the non-structured domains only. This unexpected way of crosslinking operated in chromatin, H1-depleted chromatin and core particles, i.e. independently of the chromatin structure. The efficiency of crosslinking, however, showed such a dependence: whilst the yield of crosslinks was similar in total and H1-depleted chromatin, in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. The decreased crosslinking efficiency, especially dramatic in the case of H3, is attributed to a reduced number of binding sites, and, respectively, is considered as a direct evidence for interaction of nonstructured tails of core histones with linker DNA.

Animals↗
PubMed1989

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking.

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA.

Acetylation↗
PubMed1988

Protein-DNA crosslinking in reconstituted nucleohistone, nuclei and whole cells by picosecond UV laser irradiation.

A picosecond UV laser was used to cross-link proteins to DNA in nuclei, whole cells and reconstituted nucleohistone. Irradiation of the nucleohistone resulted in crosslinking 15-20% of bound histones to DNA in a very short time (one or several picosecond pulses), the efficiency of crosslinking to single stranded DNA being higher than to double stranded DNA. All histones as well as high mobility group 1 proteins were identified in the covalently linked protein-DNA complexes upon irradiation of isolated nuclei and whole cells. A method is suggested for isolation of crosslinked material from cells and nuclei in amounts sufficient for further analysis. Experiments with reconstituted nucleohistones showed that upon irradiation at a constant dose the efficiency of crosslinking depended on the intensity of the light, thus suggesting a two-quantum process is involved in the reaction.

Animals↗
PubMed1987

Accessibility of the globular domain of histones H1 and H5 to antibodies upon folding of chromatin.

Antibodies to the globular domain of histones H1 and H5 were purified by affinity chromatography and used to study the accessibility of this region of H1 and H5 in folded and unfolded rat liver and hen erythrocyte chromatin respectively. The different conformations of the chromatin filament were induced by varying the ionic strength from 1 mM to 80 mM NaCl and maintained by fixation with glutaraldehyde. Treatment with glutaraldehyde at a given salt concentration affected neither the orientation of nucleosomes relative to the fiber axis nor the compactness of chromatin. Solid-phase immunoassay and inhibition experiments showed no binding of the antibody against the globular domain of H1 to chromatin at the entire range of salt concentrations, while the antibody to the whole H1 molecule reacted with chromatin at low salt. On the other hand, the antibody to the globular region of H5 reacted with hen erythrocyte chromatin independently of the extent of chromatin condensation. These results indicate that the antigenic determinants of the globular domain of H5 are accessible to the antibody both in folded and unfolded chromatin, while those of the same region of H1 are masked, probably by interaction with DNA or proteins.

Animals↗
PubMed1987

The globular domain of histone H5 is internally located in the 30 nm chromatin fiber: an immunochemical study.

The location of the globular domain of histone H5 relative to the axis of the 30 nm chromatin fiber was investigated by following the accessibility of this region of the molecule in chicken erythrocyte chromatin to specific antibodies as a function of chromatin structure. Antibodies to the globular domain of H5 as well as their Fab fragments were found to react with chromatin at ionic strengths ranging from 1-80 mM NaCl, the reaction gradually decreasing upon increase of salt concentration. If, however, Fab fragments were conjugated to ferritin, no reaction of the complex with chromatin was observed at salt concentrations higher than 20 mM. The accessibility of the globular part of H5 in unfolded chromatin to the Fab-ferritin complex was also demonstrated with trypsin-digested chromatin. The experiments were carried out by both solid-phase immunoassay and inhibition experiments. The data obtained are consistent with a structure in which the globular domain of H5 is internally located in the 30 nm chromatin fiber.

Animals↗
PubMed1986

Chromatin superstructure. A study with an immobilized trypsin.

Hen erythrocyte chromatin was treated with trypsin immobilized on collagen membranes and the unfolding of chromatin fiber was followed by light scattering at 90 degrees and flow linear dichroism. Chromatin was found almost completely decondensed when the bulk of H1 and H5 was digested while H3 was still intact. Further digestion leading to degradation of both H3 and the rest of H1 and H5 accounted for no more than 10-15% of the total effect. When chromatin with trypsin-cleaved H1 and H5 was titrated with increasing amounts of spermidine it folded similarly to the control sample. This finding suggests that charge neutralization appears a likely mechanism for maintaining the structure of the 30 nm chromatin fiber by the C-terminal domain of H1 and H5.

Animals↗
PubMed1984

The role of histone H1 and non-structured domains of core histones in maintaining the orientation of nucleosomes within the chromatin fiber.

Calf thymus chromatin was digested with trypsin and the structural alterations which occurred were followed by flow linear dichroism. After a sharp initial increase, the amplitude of the positive signal gradually decreased followed by a change of the sign of the dichroism and further increase of the negative signal up to a plateau. These changes of the dichroism were compared to the respective changes in the histone pattern. It was shown that the positive dichroism of chromatin did not depend on the condensation state of chromatin, and that the orientation of the nucleosomes along the chromatin fiber was maintained by the globular domain of H1 and the non-structured parts of core histones.

Animals↗
PubMed1983

Histone acetylation in chromatin containing mouse satellite DNA.

Histone acetylation in transcriptionally inactive chromatin has been studied with chromatin containing mouse satellite DNA. The latter was obtained by digestion of nuclei from Ehrlich ascites tumor cells with the restriction nuclease Bsp, which degrades main-band DNA but leaves satellite DNA intact. The enzyme-resistant material was separated by gel filtration. Satellite DNA amounted to 65% of the total DNA in this fraction. When the cells were grown in the presence of sodium n-butyrate to inhibit histone deacetylation, a few, if any, hyperacetylated forms of core histones were found in satellite chromatin. Conversely, the highest quantity of tetraacetylated H4 molecules was found in the fractions containing the most extensively degraded chromatin.

Acetylation↗
PubMed1981

Studies on the thermal denaturation of histone-H1-depleted chromatin.

The multiphasic thermal denaturation profile of histone-H1-depleted chromatin was studied by using a nucleoprotein preparation which lacked the first high temperature transition at about 72 degrees C. Such a preparation was obtained by heating at 72 degrees C H1-depleted chromatin, the DNA of which was cross-linked with psoralen to ensure a complete renaturation of DNA upon cooling. When this nucleoprotein was redenatured, its melting profile was found to be significantly altered: only one high temperature peak centered at about 82 degrees C was observed in addition to the low temperature transition at about 53 degrees C. The kinetics of digestion of this material with micrococcal nuclease showed a limit digest equal to that found for the 'native' H1-depleted chromatin but the rate of hydrolysis was higher. The monomer particles prepared from this nucleoprotein were found similar to the 'native' monosomes in respect to protein:DNA ratio and size of DNA but showed an altered melting profile: the premelt area was broader, bigger, and centered at lower temperature; the main peak was reduced in size with no change in its melting temperature. On the basis of these data, the following conclusions were drawn: (a) the last two thermal transitions in H1-depleted chromatin most likely reflect the presence within each nucleosome of two regions with different stability of DNA; (b) DNA involved in the first high thermal transition of H1-depleted chromatin belongs to nuclease-accessible DNA, and (c) the main peak in the biphasic melting profile of the monomer particles reflects the denaturation of DNA regions which in the polymer nucleoprotein are involved in the two high temperature transitions.

Animals↗