A study was done regarding the effect of the oxidizing agent potassium chromate (K 2 CrO 4 , PC) on cultured dermal fibroblasts of a healthy donor and three patients with rheumatoid arthritis (RA). Characteristics of the rRNA gene (RG) complex—RG copy number, active RG (ARG) dosage, and 18S rRNA content—were determined for each cell line. In cells of the healthy donor, oxidative stress caused by low doses of PC (2–4 µM, 1–4 h) induced an early response, including a 50–80% increase in total RNA and rRNA. An appreciable activation of the nucleolus was observed cytochemically, by silver staining and morphometry. The early response grew considerably lower with the increasing passage number and/or PC concentration. Exposure to 6–12 µM PC for 24 h led to a progressively increasing cell death rate (late response). The existence and intensity of the early response correlated positively with cell survival during further culturing. Cells of the RA patients displayed almost no early response even at early passages: total RNA did not increase, and rRNA increased by no more than 10%. Cell disruption (apoptosis) during further culturing was more intense than in the line originating from the healthy donor. The apoptosis intensity characterized by the increase in the content of DNA fragments in the culture medium and in the caspase 3 activity was inversely proportional to the ARG dosage in the genome. The results provide the first quantitative characterization of the early and late responses of cells to PC-induced oxidative stress and suggest the role of ARG dosage in cell survival during stress.

The most important stage in the making of mutations is a reparation of different DNA damage, including the more deleterious double-strand DNA breaks (DSB). The first stage of adaptive response--fundamental antimutagenic cell reaction, purposeful to reparation for induced DSB repair--is investigated in present work. Non-radioactive in situ hybridization of biotin-labeled DNA probe was used to mark chromosome 1 pericentromeric regions (PR) in G0 human lymphocytes. It was shown that under 3-10 cGy (X-radiation, 160 kV) PR become displaced from a nucleus periphery to inner territory of a nucleus. The moving process realizes during several hours after an irradiation. As far as some non-specific gene repressors are co-localized with chromosome centromeric regions it is possible hypothesizes that the displacement cause changing expression of some genes. It is possible to propose that an absence of radiation induced chromosome locus displacement may be one of causes DSB repair disturbance. This hypothesis was tested by the model. It is assumed that one consequence of the underlying defect may be inappropriate involvement of cell's recombination machinery in the repair of DSB. We studied lymphocytes of patients with hereditary BRCA2 mutation. It is thought that this gene takes part in DSB repair. The significant differences of the PR moving between control samples and the cases were revealed under 10 cGy. Similar results were observed on lymphocytes of patients with Fanconi syndrome. Thus, abnormal moving of interphase nucleus chromosomes conditioned by low-dose irradiation may suggest on imperfect machinery of DSB repair, i.e. genetic risk. We realize that further investigations are needed for definitive conclusion.

Dynamics of telomere sequences are considered in normal and immortalized cells. Immortalized cells are suggested to be derived mainly from a special subpopulation of ontogenetic reserve cells, Their epigenetic program consists of autoregeneration during external stimulus for genome reorganization and corresponding appearance of generic variants of cells. It is suggested that cells surviving the crisis stage contain a special signal sequence integrated into telomere DNA. Its elimination during shortening of DNA telomere sequences in dividing ontogenetic reserve cells is a signal for the cooperative transition of chromatin into a new steady state that corresponds to the epigenotype of immortalized cells. Localization of telomere DNA sequences in intrachromosomal "hot spots" reflects phylogenetic rearrangement of the genome.

The model is based on the concept of programmed initiation of genetic damage in sub-populations of specific evolutionary reserve cells (ERC). The model quantitatively predicts a dose response of genetic lesions at low dose range and furnishes an explanation of the minimum observed in the dose-response curve at doses corresponding to one (on the average) event of energy deposition per ERC. The complex shape of the dose-response curve is demonstrated to result from superposition of processes in different sub-populations within the exposed cell population (at low doses mainly in ERC). Programmed initiation of genetic lesions in ERC requires two hits to cell membrane and probably, at the same time, to the cell nucleus. The equation for dicentric yield in human lymphocytes as a function of dose describes the experimental observations rather well.

The dependence of UV-induced unscheduled DNA synthesis (UDS) in the unstimulated lymphocytes of human peripheral blood on the ionic strength (mu) of the culture medium has been shown. In the level of mu lower or higher than the physiological (mu(ph)) one, UDS significantly decreases. The effect of modification of mu due to the changes of ionic strength is absent in the lymphocytes of the classic form of xeroderma pigmentosum. The phenomenon may become useful in the development of a new test for revealing cells with a genetically or physiologically changed system of UV-induced DNA repair. The mechanisms of investigated phenomenon, particularly their dependence on the structure of chromatin, as well as the influence of ionic strength on binding of the repair enzymes with DNA are discussed.

The dependence of UV-induced unscheduled DNA synthesis (UDS) in non-stimulated lymphocytes of human peripheral blood on the ionic strength (mu) of the culture medium has been shown. With the level of mu lower or higher than physiological (mu(ph)) the UDS significantly decreases. The effect of modification of mu due to changes in ionic strength is absent in the lymphocytes of patients with the classic form of xeroderma pigmentosum. This phenomenon may become useful for development of a new test revealing cells with genetically or physiologically changed system of UV-induced DNA repair. Mechanisms of investigated phenomenon, particularly their dependence on the chromatin structure, as well as the influence of ionic strength on binding the repair enzymes with DNA are discussed.

A probe containing full-size DNA copy of influenza A/USSR/90/70 virus protein gene M labeled with biotin on 32P was used for influenza A virus RNA detection by dot hybridization method. For labeling with biotin, a new method of its administration by chemical modification of nucleic acid was employed. In homologous DNA:DNA hybridization the sensitivity of determinations was less than 1 pg in the biotin-treatment of the probe and 1.25 pg in its radioactive labeling. Hybridization of DNA probe with cytoplasmic RNA isolated from influenza A virus-infected (strains A/USSR/90/77 and A/Texas/77) MDCK cells revealed RNA in the dot corresponding to 4.5-5.5 1g ID50 of virus present in 2 x 10(4) cells. The probe did not bind with negative controls in any dot in all the tests. The results of the study indicate that DNA probes labeled with biotin and 32P and used in dot hybridization for influenza A virus RNA detection in infected cells show the similar sensitivity and specificity.

We have studied the changes in uv absorption and ORD in early and late stages of the reaction of formaldehyde with DNA inside Sd and T2 phages. The 20-to 25-min incubation of phages with formaldehyde at temperatures of about 40°C does not disrupt the phage particles but nevertheless causes the development of a typical hypochromic effect reaching 10±1% at 260 nm and similar to the hypochromism that develops during disruption of the phage in a medium without HCHO. The complete additivity is observed between the hypochromism at 260 nm arising as a result of incubation with HCHO and the hypochromic effect produced by disruption of the phage. The development of hypochromism under these conditions is accompanied by restoration of the positive peak of rotation with a maximum at 289 nm, characteristic of native DNA in solution and greatly reduced in intraphage DNA. The observed effects are interpreted as a conformational transition—a restoration of the Watson-Crick type of structure in the part of DNA whose secondary structure was changed in situ due to interaction with protein. All these optical changes of intraphage DNA under the influence of formaldehyde are reversible. The removal of HCHO leads to restoration of the anomalous optical properties of intraphage DNA, including deficit of hypochromism and a sharp decrease in positive rotation in the region of 280–290 nm. The rate constants, energy of activation, and entropy of activation for the reactions of formaldehyde with a mixture of bases, with free native and intraphage DNA were calculated.