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A N Luchnik

Publications and source records attributed to A N Luchnik.

At least 19 recordsLinked to original sources
PubMed1991

A. E. Braunstein Plenary Lecture. Nuclear skeleton, DNA domains and control of replication and transcription.

Chromosomal DNA is organized in loops or domains of about 100 kb. Their ends seem to be attached to special protein skeletal structures. The DNA-attachment sites can be subdivided into permanent and transient types. The permanent or constitutive attachment sites, which are retained in all types of cells (including those inactive in replication and transcription), either coincide with or are located close to replication origins. This observation provides a simple way for isolation of DNA fragments containing replication origins. Such fragments from the chicken alpha-globin gene domain and other regions of the chicken genome contain DNA sequences which interact with nuclear proteins present in dividing cells, but absent from non-dividing cells. Several new consensus sequences interacting with nuclear proteins were detected. The 5' end region of the alpha-globin gene domain containing a replication origin was found to possess enhancer activity lacking tissue specificity. Hence, the domain organization of DNA is related to the organization of replication process. Other sets of data indicate that the integrity of DNA domains is important for maintaining transcription within the domain. According to these data, even a single nick at an distance of about 100 kbp seems to be sufficient for blocking transcription within the whole domain at the stage of RNA elongation. Thus, topological integrity of DNA may be an important factor involved in formation of active chromatin.

Animals↗
PubMed1991

Relationship of histone acetylation to DNA topology and transcription.

An autonomously replicating plasmid constructed from bovine papiloma virus (BPV) and pBR322 was stably maintained as a nuclear episome in a mouse cell culture. Addition to a cell culture of sodium butyrate (5 mM) induced an increase in plasmid DNA supercoiling of 3-5 turns, an increase in acetylation of cellular histones, and a decrease in plasmid transcription by 2- to 4-fold. After withdrawal of butyrate, DNA supercoiling began to fluctuate in a wave-like manner with an amplitude of up to 3 turns and a period of 3-4 h. These waves gradually faded by 24 h. The transcription of the plasmid and acetylation of cellular histones also oscillated with the same period. The wave-like alterations were not correlated with the cell cycle, for there was no resumption of DNA replication after butyrate withdrawal for at least 24 h. In vitro chemical acetylation of histones with acetyl adenylate also led to an increase in the superhelical density of plasmid DNA. The parallel changes in transcription, histone acetylation, and DNA supercoiling in vivo may indicate a functional innerconnection. Also, the observed in vivo variation in the level of DNA supercoiling directly indicates the possibility of its natural regulation in eukaryotic cells.

Acetylation↗
PubMed1991

High rotational mobility of DNA in animal cells and its modulation by histone acetylation.

DNA rotational mobility in a bovine papilloma virus (BPV)-based minichromosome, autonomously replicating in mouse cells, was studied using topoisomer analysis in temperature shift experiments. It was found that in live cells the average number of topological turns increased by six in the course of temperature shift through a range of 37 degrees C. This comprised approximately 85% of the total potential mobility of naked plasmid DNA. DNA rotation in isolated nuclei was found to be 3.5-4.0 turns per 37 degrees C in 100 mM NaCl - much higher than in all experiments with animal cells reported thus far. In low salt mobility was considerably lowered. Attempts to extract minichromosomes from nuclei allowed isolation of no more than 10% of minichromosomal DNA, with could indicate a very high proportion of transcriptionally active minichromosomes in the intracellular population. Growing cells in the presence of sodium butyrate resulted not only in an increase in the level of plasmid superhelicity and a decrease of its transcription (as we report in the accompanying publication) but also reduced rotational mobility of plasmid DNA threefold (from 6 to 2 turns per 37 degrees C). The decrease in DNA rotational mobility after butyrate treatment was also partially manifested in isolated nuclei (especially at lower ionic strength). To check whether histone acetylation is directly responsible for DNA immobilization, we performed in vitro acetylation of histones using acetyl adenylate. This resulted in severe DNA immobilization in experiments using both up and down temperature shifts.

Acetylation↗
PubMed1988

Inhibition of transcription in eukaryotic cells by X-irradiation: relation to the loss of topological constraint in closed DNA loops.

X irradiation was found to inhibit in vivo transcription in mammalian, yeast, insect and avian cells in a dose-dependent manner. Measurements of DNA nicking indicated that about one DNA single-strand break per estimated DNA loop (domain) length is sufficient to explain the effect. The inhibitory effect was partially reversed by post-irradiation incubation of cells. During such incubation DNA nicking was considerably repaired. The size of transcripts was not changed by irradiation. The in vitro (run on) activity of RNA polymerase in nuclei isolated from irradiated cells also was not altered. The dose-response curves were different in various cells, correlating with the reported unequal average domain size of supercoiled DNA (and also replicon size) in diverse organisms.

Chromatin↗
PubMed1985

Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA.

The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl-containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA.

Chromatin↗
PubMed1985

DNAaseI-hypersensitive minichromosomes of SV40 possess an elastic torsional strain in DNA.

Previously, we have shown that DNA in a small fraction (2-5%) of SV40 minichromosomes was torsionally strained and could be relaxed by treating minichromosomes with topoisomerase I. This fraction was enriched with endogeneous RNA polymerase II (Luchnik et al., 1982, EMBO J., 1, 1353). Here we show that one and the same fraction of SV40 minichromosomes is hypersensitive to DNAase I and is relaxable by topoisomerase I. Moreover, this fraction completely loses its hypersensitivity to DNAase I upon relaxation. The possibility that this fraction of minichromosomes can be represented by naked DNA is ruled out by the results of studying the kinetics of minichromosome digestion by DNAase I in comparison to digestion of pure SV40 DNA and by measuring the buoyant density of SV40 chromatin in equilibrium CsCl gradient. Our data obtained with SV40 minichromosomes may be relevant to the mechanism responsible for DNAase I hypersensitivity in the loops or domains of cellular chromatin.

Animals↗
PubMed1984

[Phosphorylation of the nuclear matrix proteins of the liver and of Zajdela's hepatoma in the rat].

The rate of protein phosphorylation in isolated nuclear matrices from liver and Zajdela's hepatoma is very high, being even slightly higher than in isolated nuclei. This indicates that active protein kinases remain tightly bound to the nuclear matrix, since the areas of phosphorylation are the same. However, during isolation of the nuclear matrix from labeled nuclei in the absence of proteolysis and dephosphorylation inhibitors, most part of the label is eliminated from nuclear matrix proteins. These proteins were phosphorylated in both the tissues, however, in Zajdela's hepatoma, two proteins with a molecular weight of 29-31 kD and four proteins with a molecular weight of 12-19 kD were phosphorylated more intensely than in the liver.

Animals↗
PubMed1983

[Disorder of the repair of DNA double-stranded breaks in radiosensitive mutants of Saccharomyces cerevisiae yeasts].

DNA double-strand break repair and restoration of viability in X-irradiated diploid yeast cells homozygous for rad50, rad51, rad52, rad55 mutations were studies under conditions of keeping the cells in non-nutrient medium, after irradiation. All the cells were synchronized at the G1 stage of the cell cycle. In contrast to the wild-type yeast, this group of mutants are unable to repair DNA double-strand breaks and do not enhance viability, when kept in non-nutrient medium after irradiation.

DNA Repair↗
PubMed1983

[Changes in DNA supercoiling during differentiation, aging and malignant transformation].

The data on changes in DNA supercoiling during cell differentiation, in vitro aging and SV40 virus-induced or spontaneous malignant transformation are discussed. The observed correlation between the level of cell proliferation and the density of DNA topological ("titratable superhelical") turns in the closed nuclear DNA loops, as well as the data on the effect of DNA supercoiling on transcription allow to suggest that the selective and co-ordinated switch in the transcription of genes involved in the control of cell proliferation may operate through total changes in the level of supercoiling of the closed nuclear DNA loops.

Animals↗