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Biomedical subjects

N D Kobets

Publications and source records attributed to N D Kobets.

At least 19 recordsLinked to original sources
PubMed1999

Formation of nucleosomes does not suppress interaction of a DNA fragment with an alkylating derivative of a pyrimidine oligonucleotide.

Oligonucleotide derivatives capable of binding to specific nucleic acids are considered as potential therapeutic agents, exerting their action at the level of genome functioning (Hélène, 1991; Knorre et al., 1993). A straightforward approach to targeting DNA is based on using oligonucleotides capable of binding to oligopurine-oligopyrimidine sequences by formation of triple-strand structures. We report results of experiments on sequence-specific chemical modification of a 490-bp fragment of pfosCAT plasmid, containing the promoter segment of the c-fos gene using 4-(N-2-chloroethyl-N-methylamino)-benzylphosphamide derivatives of a homopyrimidine 14-mer oligonucleotide. It was shown that in both the free DNA and the DNA involved in nucleosome structure, reaction occurred with similar efficiency at the target guanosine residue G404.

Alkylation↗
PubMed1998

Chromatin proteins surrounding the d(G-T)n repeats and polyamine influence as revealed by photoaffinity labeling with reactive pd(A-C)6 derivatives.

The complementary-addressed modification of DNA and proteins in chromatin using photoreactive derivatives of pd(AC)6 has been studied. These oligonucleotides form complementary complexes with specific DNA sequences and modify both DNA and proteins in the vicinity of these regions, and can be used for investigation of the protein environment in DNA. We have demonstrated that photoreactive derivatives of oligonucleotides can quickly and efficiently modify chromatin proteins and seem to be promising for investigation of perturbations in chromatin structure during the cell cycle. A comparison between modified chromatin from synchronized cells has demonstrated differences in the sets of proteins modified in the S and G1/S phases of the cell cycle. An increase in spermine and spermidine concentrations leads to an increase in modification of definite chromatin proteins. It can be supposed that the B-Z transition that can be stabilized by the presence of natural polyamines is one of the reasons for the presence of single-stranded DNA regions, containing sets of (dG-dT)n and accessible for interaction with complementary oligonucleotides.

Cell Nucleus↗
PubMed1998

[Chemical modification of eukaryotic cell chromatin, directed at d(GT)n-repeats of DNA].

DNA and proteins of chromatin from eukaryotic cells were specifically modified by an alkylating derivative of pd(AC)6 (complementary to d(GT) repeats of DNA) containing a 4-(N-methyl-N-2-chlorethylamino)benzylamine residue on its 5'-end. It was shown that the efficiency of modification of both DNA and proteins increases in the presence of spermine and spermidine and sharply decreases after preliminary treatment of chromatin by nuclease S1 under conditions of mild cleavage of single-stranded DNA regions. It was suggested that one of the reasons for the presence of unwound d(GT)n stretches in chromatin DNA accessible for interaction with the complementary oligonucleotide is the B-->Z transition. Proteins specifically alkylated within the chromatin, which most likely are located in the regions of local unwinding of DNA, near the repeats, were analyzed.

Animals↗
PubMed1994

Oligo(A), oligo(TG), and Alu repeats of DNA in chromatin are available for sequence-specific chemical modification with oligodeoxynucleotide derivatives.

Reaction of 4-(N-2-chloroethyl-N-methylamino) benzylphosphamides of oligonucleotides, which are targeted to the poly(A), poly(TG), and Alu repeats of eukaryotic DNA in chromatin and isolated nuclei from HeLa cells, has been investigated. It was found that the reagents alkylate DNA and some proteins due to specific complex formation. The affinity character of the reaction was proved by the fact that free corresponding oligonucleotides taken in excess or preliminary treatment of chromatin with S1 nuclease both prevent the biopolymers from the modification. Deproteinated DNA from the same cells does not react with oligonucleotide derivatives. This suggests that the chromatin DNA must have some structural features allowing oligonucleotide binding. Reactivity may be attributed to the existence of strongly negative supercoiled DNA regions containing single-stranded sequences or regions where DNA can unwind in the presence of complementary oligonucleotides. Results obtained suggest that in eukaryotic chromatin there are open DNA sequences available for affinity modification with oligonucleotide derivatives not only due to formation of triple helixes.

Adenine Nucleotides↗
PubMed1993

[Interaction of photoactive oligothymidylate derivatives with HeLa cell chromatin].

Photoactive derivatives of d(pT)16, bearing arylazide, nitroarylazide and perfluoroarylazide residues, were used for the complementary addressed modification of DNA and proteins in chromatin. As compared with alkylating derivatives, the photoactive compounds possess higher efficiency and specificity, and shorter incubation times which prevents nucleus from degradation. These reagents can therefore be used for identification of proteins located near to particular DNA regions in chromatin.

Azides↗
PubMed1993

[Synthesis of oligodeoxyribothymidylate derivatives containing alkylating groups and residues of biotin, for directed modification of chromatin].

An oligodeoxythymidylate derivative bearing an alkylating group at the 5'-end and biotin at the 3'-end (RCl-(pdT)16-bio) was synthesized. This reagent alkylates polyA-tracts of DNA in HeLa nuclei specifically via complementary complexes with single-stranded segments of DNA and by the preliminary treatment of chromatin with S1-nuclease, since the reaction is inhibited by an excess of the corresponding free oligothymidylate. The reagent will be used to study the distribution of local unwinded parts of polyA-repeats of DNA in human chromatin by the electron microscopy.

Alkylating Agents↗
PubMed1992

Affinity modification of human chromatin with reactive derivatives of oligonucleotides.

Reaction of 4-(N-2-chloroethyl-N-methylamino)benzylphosphamides of oligonucleotides (RCl-(pT)16 and RCl-(pApC)6) with human chromatin in intact nuclei and with metaphase chromosomes has been investigated. The oligonucleotides were targeted to poly(A) and poly(TG)-repeating DNA sequences. It was found that the reagents alkylate DNA and some proteins due to specific complex formation. The affinity character of the reaction was proved by the fact that free corresponding oligonucleotides taken in excess or preliminary treatment of chromatin with S1-nuclease both prevent the biopolymers from modification. The results obtained evidence that in human chromatin there are open DNA sequences available for affinity modification with oligonucleotide derivatives. Analysis of patterns of modified proteins within these chromatin areas may give a key to the structure of these chromatin sites.

Benzene Derivatives↗
PubMed1991

[Chromatin proteins in rat liver cells, interacting with reactive oligothymidylate derivatives].

Upon alkylation of the rat liver chromatin with a hexadecadeoxyribothymidylate derivative bearing 4-(N-2-chloroethyl-N-methylamino)benzylamine residue on the 5'-terminal phosphate two nonhistone proteins were modified under conditions of the reagent's forming complementary complexes with poly(A) sequences in DNA. The sequence-specific nature of the reaction is proved by the competition experiments: free oligothymidylate prevented the proteins from alkylation whereas arbitrary oligonucleotide did not. Modification with the reactive oligonucleotide derivatives can be used for the identification of proteins located in the vicinity of the specific open DNA regions in chromatin.

Alkylation↗
PubMedDate not supplied

[Synthesis, properties and interaction with eukaryotic cells of alkylating derivatives of oligodeoxyribonucleotides containing cholesterol or phenazinium residue covalently bound to the 3'-terminus].

Radioactive alkylating 5'-[32P]-[4-(N-2-chlorethyl)N-methylaminobenzyl]-5'-phospham ide decadeoxyribothymidilate derivatives containing either free hydroxyl group (reagent I), hydrophobic cholesterol residue (reagent II) or polyaromatic phenazinium residue (reagent III) at 3'-termini were synthesized. The products were purified by HPLC and used for oligonucleotide-directed alkylating of DNA in isolated rat liver nuclei, Krebs-2 ascite carcinoma cells and L-929 murine fibroblasts. The uptake of reagent II by the cells was two orders of magnitude higher than that of reagent I and III. Intracellular alkylation of DNA by reagent II both in isolated nuclei and in living cells was about one order of magnitude higher than in the case of reagent I. The presence of phenazinium at 3'-termini of the reagent III leads to a sufficient increase of the alkylation extent compared to reagent I despite a quite low extent of its uptake by the cells.

Alkylating Agents↗
PubMedDate not supplied

[Affinity modification of Escherichia coli ribosomes in the region of the mRNA-binding center by a heptauridylate analog bearing a chemically active group at the 3' end].

4-(N-2-chloroethyl-N-methylamino)-benzaldehyde acetyl derivative (RCL-derivative) of hepatauridylic acid was used to localize the structure organizing the mRNA-binding site of ribosomes. This derivative; like a free oligonucleotide, stimulates the binding of [14C]phenylalanyl-tRNA to ribosomes and effectively alkylates ribosomes, mainly the 30S subunit within the specific complex. The alkylation being completely inhibited by preincubation with polyuridylic acid, suggests that the chemical alteration occurs near the mRNA-binding site. Both rRNA and proteins undergo modification in the 30S subunit (15 and 85% of the total 30S subunit, respectively). The radioactive marked was found in fractions of proteins S18, S9 and S1.

Affinity Labels↗