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This paper presents the principles and application of DNA based biological UV dosimeters, as developed by Research Group for Biophysics (RGB). These dosimeters are used for assessing the biological hazard of living systems on the Earth's surface and in different waters (rivers, lakes, seas, etc.). The UV dosimetry system has also been used in the space. In dosimeters a bacterial virus, bacteriophage T7 and polycrystalline uracil thin layers have been used as biological detectors. On the Earth's surface the UV radiation induces dimer formation in phage T7 and in the uracil detector, which was evaluated by loss of viability of the phage particles and by the decrease of the characteristic optical density (OD) of uracil thin layers. Recently the development of human space activities has also increased the need to measure the biological effect of extraterrestrial solar radiation, too. The evaluation of the space samples occurred on ground, thus only the starting and the final state were taken into account. A new improved, automated method is presented below which makes data collection more efficient and also makes the dynamics of the process observable.

The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522(T) was exposed to UV doses over a wavelength range of 200-400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m(2), and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m(2). The estimated D(37) (dose of 37 % survival) for Hcc. dombrowskii was > or = 400 kJ/m(2). However, exposure of cells to UV flux while in liquid culture reduced D(37) by 2 orders of magnitude (to about 1 kJ/m(2)); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.

Polycrystalline uracil thin layer can be used as biological dosimeter for assessing exposure to UV radiation. The dimerization and reversion efficiency of the ultraviolet radiation in the UV-B and the UV-C range were quantified on polycrystalline uracil thin layers irradiated with quasi-monochromatic radiation using interference filters of 10nm bandwidth. The dimer formation and monomerization (reversion) dose-effect relations were determined by optical spectroscopy. The decrease of the OD value of the uracil thin layer at 288 nm was taken as a measure of the dimer formation, while the increase of the OD of a completely irradiated (until reaching the saturation level) uracil layer was taken as the sign of the monomerization. The two processes in the UV-B and the UV-C range take place simultaneously, the individual characterization of the dimerization efficiency was performed from the initial slope of the dimerization dose-effect function and an action spectrum for dimerization was constructed in the UV-C range too. The reversion efficiency was found to be practically the same with all of the investigated wavelengths: 200 nm, 210 nm, 220 nm, 230 nm, 240 nm The possible biological relevance of the reversion of dimers are discussed.

The experiment "Phage and Uracil Response" (PUR) will be accommodated in the EXPOSE facility of the International Space Station (ISS). Its objective is to examine and quantify the effect of specific space conditions on bacteriophage T7 and isolated T7 DNA thin films. In order to define the environmental and technical requirements of the EXPOSE, the samples were subjected to the Experiment Verification Test (EVT). During EVT the samples were exposed to selected space conditions: high vacuum (10(-4) to 10(-6) Pa) and UV-C radiation (254 nm) alone and in combination. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA/phage and the decrease of the amount of PCR products have been detected indicating the damage of isolated and intraphage T7 DNA. Intraphage DNA is more sensitive to simulated space parameters than isolated T7 DNA in thin layers as well. We obtained substantial evidence that DNA lesions accumulate throughout exposure, and the amount of damage depends on the thickness of the layers. According to our preliminary results, the damages by exposure to conditions of dehydration and UV irradiation are larger than the sum of vacuum alone, or radiation alone case, suggesting a synergistic action of space vacuum and UV radiation with DNA being the critical target.

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m(2)) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m(2), differences between these tests were observed. Antioxidant effect was modeled by alpha-tocopherol-acetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m(2)), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.

Ultraviolet B (UV-B) radiation is a modality widely used for the treatment of different skin diseases. One of the major mechanisms of UV-B immunosuppression in this treatment modality is thought to be an apoptosis-inducing effect on T cells infiltrating the skin. We examined the T-cell apoptosis-induction capacities of four different UV light sources, with and without UV filters. The xenon chloride (XeCl) laser proved to be the strongest apoptosis inducer. The use of a phtalic acid filter eliminated UV radiation almost completely below 300 nm, which resulted in a severe decrease in the apoptosis-inducing capacity of different UV-B sources. Using the results of the measurements with polychromatic UV light sources, the wavelength dependence of UV-B light for the induction of T-cell apoptosis was also determined. The regression line of the action spectrum demonstrated a continuous decrease from 290 to 311 nm. The apoptosis-inducing capacity of the XeCl laser was almost four times higher than the calculated value according to the action spectrum, which might be attributed to the high irradiance of the laser as compared with nonlaser light sources.

Polycrystalline uracil thin layers participate in the phage and uracil response (PUR) experiment, assigned to the biological dosimetry of the extraterrestrial solar radiation on the International Space Station (ISS). In ground based experiments (experiment verification tests), the following space parameters were simulated and studied: temperature, vacuum and short wavelength UV (UV-C, down to 200 nm) radiation. The closed uracil samples proved to be vacuum-tight for 7 days. In the tested temperature range (from -20 to +40 degrees C) the uracil samples are stable. The kinetic of dimer formation (dimerization) and reversion (monomerization) of uracil dimers due to short wavelength UV radiation was detected, the monomerization efficiency of the polychromatic deuterium lamp is higher than that of the germicidal lamp. A mathematical model describing the kinetic of monomerization-dimerization was constructed. Under the influence of UV radiation the dimerization-monomerization reactions occur simultaneously, thus the additivity law of the effect of the various wavelengths is not applicable., (c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.)

The ultraviolet (UV) environment of Mars has been investigated to gain an understanding of the variation of exposure throughout a Martian year, and link this flux to biological effects and possible survival of organisms at the Martian surface. To gain an idea of how the solar UV radiation varies between different regions, including planned landing sites of two future Mars surface missions, we modelled the total solar UV surface flux throughout one Martian year for two different dust scenarios. To understand the degree of solar UV stress on micro-organisms and/or molecules essential for life on the surface of Mars, we also calculated the biologically effective dose (BED) for T7 and Uracil in relevant wavelength regions at the Martian surface as a function of season and latitude, and discuss the biological survival rates in the presence of Martian solar UV radiation. High T7/Uracil BED ratios indicate that even at high latitudes where the UV flux is significantly reduced, the radiation environment is still hostile for life due to the persisting UV-C component of the flux., (c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.)

The main goal of PUR experiment (phage and uracil response) is to examine and quantify the effect of specific space conditions on nucleic acid models. To achieve this an improved method was elaborated for the preparation of DNA and bacteriophage thin films. The homogeneity of the films was controlled by UV spectroscopy and microscopy. To provide experimental evidence for the hypothesis that interplanetary transfer of the genetic material is possible, phage T7 and isolated T7 DNA thin films have been exposed to selected space conditions: intense UVC radiation (lambda=254 nm) and high vacuum (10(-4) Pa). The effects of DNA hydration, conformation and packing on UV radiation damage were examined. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA and the decrease of the amount of PCR products have been detected indicating the photodamage of isolated and intraphage DNA., (c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.)

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.

The UV radiation environment on planetary surfaces and within atmospheres is of importance in a wide range of scientific disciplines. Solar UV radiation is a driving force of chemical and organic evolution and serves also as a constraint in biological evolution. In this work we modeled the transmission of present and early solar UV radiation from 200 to 400 nm through the present-day and early (3.5 Gyr ago) Martian atmosphere for a variety of possible cases, including dust loading, observed and modeled O3 concentrations. The UV stress on microorganisms and/or molecules essential for life was estimated by using DNA damaging effects (specifically bacteriophage T7 killing and uracil dimerization) for various irradiation conditions on the present and ancient Martian surface. Our study suggests that the UV irradiance on the early Martian surface 3.5 Gyr ago may have been comparable with that of present-day Earth, and though the current Martian UV environment is still quite severe from a biological viewpoint, we show that substantial protection can still be afforded under dust and ice.

Although according to the International Radiological Protection Association-International Non-Ionizing Radiation Committee recommendation (1991) the use of sunbeds for cosmetic purposes is not recommended, tanning devices are used widely. Ten different types of commercially available sunbed tubes have been studied using a uracil biological UV dosimeter, and three of them were analyzed in detail. Dimerization effectiveness of the tubes was measured directly, whereas efficiency of erythema induction was calculated weighting the emission spectra by the Commission Internationale de l'Eclairage erythema action spectrum. The data obtained demonstrate that quality control of sunbed tubes has to include not only the determination of the UV doses administered but also the assessment of the health risk due to the UVB and UVA components of the lamp. A method of quality control using the uracil biological dosimeter was elaborated, and the estimation of the "acceptable" exposure time was checked/controlled on 15 volunteers by assessing individually the erythema induction threshold. A correct classification of the sunbed tubes is proposed by characterizing the erythema induction versus DNA-damaging effectiveness of tubes.

Sunlight is the most important environmental UV source, affecting not only human health but also the whole terrestrial ecosystem. The use of artificial sources is advantageous since it is independent of geographical location and seasonal variations, however, in some photobiological/photochemical studies the choice of a specific UV source in relation to the biological end-point studied is sometimes questionable. Furthermore, it is often difficult to compare the results obtained in different laboratories due to 'slight' differences in the physical characteristics of the UV sources used. In an attempt to address these issues we calculated and compared the physical characteristics and the biological efficiency in UV-B and UV-A regions for two biological end-points (CPD and Fpg-sensitive sites formation) for frequently used UV-B, UV-A sources and solar light simulators (SLS). Our calculation shows that FS20 lamp is appropriate for studying the biological effects of UV-B radiation although differences in spectral characteristics of the associated filters may lead to at least 2-fold yields in CPD production. Furthermore, the use of a SLS with a Kodacel filter alone is inadequate for studying environmental UV effects. A metal-halide source with a Schott WG345 filter is appropriate for studies on biological effects due to UV-A region. Relative exposure duration was calculated to achieve equal amount of CPD or Fpg-sensitive sites, provided equal, total UV-(A+B) irradiance for the different UV sources., (Copyright 2002 Elsevier Science B.V.)

In order to develop monitoring and assessment systems of biologically effective doses of solar-UV radiation, concurrent measurements of spectral photometry and spore dosimetry were conducted in summer months at four sites in Japan and Europe. Effectiveness spectra were derived by multiplying spectral irradiance in 0.5 nm steps between 290 and 400 nm with the inactivation efficiency of the spores determined using monochromatic radiation of fine wavelength resolution. Shapes of the effectiveness spectra were very similar at the four sites exhibiting major peaks at 303.5, 305.0, 307.5 and 311.0 nm. The dose rates for spore inactivation from direct survival measurements and from calculations by the integration of the effectiveness spectra were compared for 174 data points. The ratios (observed/calculated) of the two values were concordant with a mean of 1.26 (+/- 0.24 standard deviation [SD]). The possible causes for the variations and slightly larger observed values are discussed.

To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage.

Biological systems used as biological dosimeters can possess different angular sensitivities from the detectors usually used in physical devices. A simple experimental setup has been developed and used to measure the angular sensitivity of uracil thin-layer biological dosimeters. Results of angular sensitivity measurements for uracil thin-layer dosimeters are presented using a Xe arc lamp as the UV source. According to the experiments described here, uracil thin-layer dosimeters show a cosine-type angular dependence. In several indoor experiments broadband UV meters are used to control the applied dose rate from a given artificial UV source. The experimental setup has been designed and used to verify experimentally the importance of spectral and angular sensitivity differences of biological and physical UV meters applied in biological experiments. Model calculations for two different irradiation systems, using different geometrical arrangements of artificial UV sources, are also presented. For these arrangements relative dose rates that could be measured with dosimeters of arbitrary spectral, but different angular sensitivity have been calculated.

Dimerization of uracil monomers in a polycrystalline state by UV radiation changes the absorption characteristics of a thin layer of the material. The change in optical density, measured by spectrophotometry in the 250-400 nm range, as a function of the exposure time is evaluated in terms of the biologically effective UV dose. A statistical evaluation of a great number of uracil dosimeters irradiated with a TL01 lamp from Philips establishes the possibility of evaluating the biologically effective UV dose using a uracil dosimeter. Nonlinear regression procedures were introduced to correct the absorption spectra for contributions due to light scattering and to determine the optical density values required to calculate the UV dose expressed in HU units. Comparison of cumulative daily doses and long-term monitoring measured by the uracil thin-layer dosimeter and a phage T7 dosimeter are given, which allow the determination of conversion factors between various biological dosimeters under different irradiation conditions.

To detect changes in DNA and/or protein structures of phage T7 under different ionic strength, Raman spectra of phage T7 have been recorded in solutions of three different NaCl + Tris concentrations. Iterative Jansson-Van Cittert deconvolution, as well as decomposition methods have been used to quantify changes in DNA structure. Significant modifications in ratios of contributions from 675 and 685 per cm vibrations, as well as in the DNA backbone vibrations, characteristic for B-DNA, near 835 per cm frequency have been found. Changes of the base electronic structure were identified in the interval between 1280 and 1400 cm(-1). Estimation of the overall protein structure suggests predominant beta-sheet content.

The dioxinocoumarin derivatives 5H-[2]benzopyrano-[3,4-g][1,4]benzodioxin-5-one (I), 5H-[2]benzopyrano-[3,4-g][2,3]-dihydro-[1,4]benzodioxin-5-on e II, 6H-[2]benzopyrano[3,4-f]-1,4-benzodioxin-6-one (III) and 6H-[2]benzopyrano[3,4-f]-2,3-dihydro-1,4-benzodioxin-6-one (IV) were synthesized. Their biological effect was studied in the presence and absence of UVA radiation, and compared with that of 8-methoxypsoralen (8-MOP) and angelicin derivatives on T7 phage, diploid yeast (Saccharomyces cerevisiae) and HeLa cells. The photobiological activities of compounds I and III were stronger than that of 8-MOP in phage inactivation and DNA synthesis inhibition in HeLa cells, whereas compounds II and IV, with a saturated dioxin ring, showed very poor activity. The photosensitizing activity of dioxinocoumarins on phage inactivation decreased by a factor of two to three in the absence of oxygen. Treatments with compound I and UVA in the presence of oxygen modified the helical structure and stability of phage DNA and proteins. Compounds I and II were more active than IV for photoinduced cell killing in yeast, although always less active than 8-MOP. At comparable photocytotoxic levels, compounds I and III were as strong inducers of cytoplasmic "petite" mutants in yeast as angelicin, suggesting a possible monofunctional mode of action with cellular DNA.

Thirty-six nitrated furan and arenofuran derivatives were measured and quantitatively characterized by the T7 inactivation test. A wide range of substances previously studied allowed us to compare the collected quantitative data with those obtained by other workers using different short-term tests. Based on comparative statistical evaluation of these data a borderline was determined for the genotoxic effect: compounds having in our short-term test mutagenicity index (MI) values smaller than 8.0 are positive while the higher values represent negative genotoxicity. Classification of 36 nitrofuran/nitroarenofuran derivatives is given both on the basis of the quantitative genotoxicity scale and in terms of +/- on the qualitative scale. All but one compound were found to be genotoxic and the genotoxic activities of these compounds were compared with the results of other carcinogenicity or mutagenicity tests.

An experimental method complete with theoretical considerations is presented for the measurement of different biological UV doses. The method is based on the high sensitivity of phage T7 activity to UV light. A precisely determined T7 inactivation action spectrum is presented over a wide optical range (240-514 nm). Using the T7 spectral sensitivity in relation to the minimal erythema dose (MED) and the effective spectral irradiance from solar radiation for the MED, an example is given to determine the MED value based on the measurement of T7 inactivation for a given case. The advantages and applicability of the method are discussed.

The irradiation of the phage T7 system containing psoralen as photosensitizer causes many processes, each of them leading to phage inactivation. These processes include the UV-induced photoreactions in the phage nucleic acid, and photoreactions in the nucleic acid sensitized by either psoralen or psoralen photobreakdown products. In addition the intercalation of the psoralen molecule itself in the phage nucleic acid as well as the psoralen photobreakdown products cause phage inactivation. Under appropriate experimental conditions these reactions can be studied and characterized separately. The quantitative characteristics (e.g. inactivation cross-section, action spectra and index for dark genotoxicity) are demonstrated for different linear and angular psoralens. Some theoretical and practical consequences of the results obtained are discussed.

The dark and photoreactions of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view. The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements. The genotoxicity of AMT in the dark is comparable with that of known genotoxic drugs as determined by phage inactivation. The photoreaction with UVA light leads to the formation of mono- and di-adducts depending on the wavelength and dose used. Mono- and di-adducts influence DNA stability differently; biologically both types of adducts are genotoxic as measured by action spectra.

Inactivation of phages T7 and phiX174 induced by the radiomimetics N-nitroso-N-methylurea (NMU) and N-acetylethylenimine (AEI) has been studied. In semilogarithmic scale the kinetic curves of phage inactivation were steplike in character. This was connected with the occurrence of several processes in the system. A possible kinetic model has been proposed for the process of NMU-induced phage inactivation. The model is applicable to initial phage inactivation only. From the model a kinetic curve parameter, rho can be determined which characterizes the radiomimetic effect on different phages and determines the changes in the repair system after caffeine treatment. NMU exerted a post-inactivation effect on the phage T7. Also the NMU-induced phage T7 post-inactivation kinetic curves were of steplike character. They can be described as a sum of two exponential processes of different rate constant. The rate constant for the first stage of the post-inactivation process was close to the depurinization rate constant found in the literature.

The authors present a method of measurement and evaluation for continuous cultures of bacteria infected by virulent phages. By the comparison of the model describing the phage-bacterium interaction and their own experimental data they determined the following biological parameter values characterizing the interaction: the adsorption rate (mu), the expectation of the latency period (T) and its standard deviation ((sigma), the time required for lysis (d), the infection efficiency (eta) and the average burst size (C). The parameters were used for the determination of optimal cultivating conditions (maximum phage yield).

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.

Structural parameters of phage T7 were compared in two frequently used Tris buffers of high and low ionic strength, in order to explain the different biological activity and drug-binding characteristics. Characteristics of the whole phage geometry were obtained by viscosimetry, static and quasi-elastic light-scattering and small-angle X-ray scattering. The latter method revealed dissimilarities in the intraphage DNA compactness, consistent with the findings of the optical absorption melting studies. Alterations in the particle dimensions determined in the same sample by different methods are discussed, and a model is constructed to explain the structural modifications that occur on lowering the ionic strength.

The action spectrum (240-300 nm) for photoinactivation of unsensitized phage T7 and the action spectra (310-380 nm) for photoinactivation of phage T7 sensitized with 8-methoxypsoralen (8-MOP) and angelicin were measured by an automated method. For unsensitized phage T7 the action spectrum is in good agreement with the absorption spectrum. For sensitization with angelicin the action spectrum is similar to the absorption spectrum, but for sensitization with 8-MOP the spectra are different. The agreement between the T7 absorption and action spectra in the far-UV region is due to photodamage of DNA, leading to phage inactivation. The similarity in the action and absorption spectra in the near-UV region for sensitization with angelicin seems to be in accordance with the monofunctional photobinding of angelicin to DNA. The action spectrum for sensitization with 8-MOP has a maximum at about 320 nm and this suggests that, in addition to the monoadducts, the biadducts play a role in the inactivation of phage T7. Taking the number of bound furocoumarin molecules into consideration, the quantum efficiencies were estimated. Furocoumarin increases the quantum efficiency in the near-UV region and the values are similar to those obtained in far-UV light without psoralens.

Optical density, fluorescence intensity, polarization and lifetime measurements were performed to analyze the temperature-induced phase transitions of phage T7 in several buffers. For labelling the intraphage DNA, ethidium bromide, proflavine and rivanol were applied for the proteins 1,6-diphenyl-1,3,5-hexatriene (DPH). In the temperature range of 20 degrees C-100 degrees C several structural changes of T7 were detected. Making corrections for light scattering by using integrating spheres and analyzing the fluorescence signals, the phase transition of intraphage DNA below 60 degrees C was interpreted as a superhelical relaxation phenomenon. The structural changes found by optical density at higher temperatures could be assigned to a change in the phage DNA or to a change in its protein part on the basis of fluorescence-melting results concerning DNA and protein labels. The effects of ionic strength and environment on the structural changes were studied.

The phenomena connected with the first phase transition step of the native T7 phage at 40 degrees-65 degrees C have been studied using various methods. In this temperature range a) the optical melting curve shows an absorption decrease, b) the maximum of the small-angle X-ray scattering characteristic for DNA packing disappears, c) there is a drop of biological activity and d) there are changes in the structure of the difference absorption spectra of native phages versus isolated DNA. All data are interpreted assuming a structural change of the DNA due to the release of its protein coat towards the end of the first phase transition step (at 60 degrees-65 degrees C in the case of M9 buffer). Above this temperature the intraphage DNA packing appears to be destroyed and the DNA structure seems to be similar to that in DNA solution.

A new method is described for the quantitative characterization of the genotoxic effect of chemicals. The method is based on the determination of the inactivation of bacteriophage T7 and on the application of a simple mathematical model valid for the processes during, or at least in the initial stage of the interaction of chemicals and phages. A value characteristic for the chemical is defined and it is determined from the inactivation kinetics. Typical inactivation kinetic curves and some problems of the application of the model as well as the mutagenicity index values determined for about 30 substances are presented. The substances examined have mutagenicity index values covering a range of six orders of magnitude. The obtained values are compared with the results of different mutagenicity/carcinogenicity tests and discussed on the basis of data in the literature. The presented method is proposed to be applied for quantitative mutagenicity screening of chemicals.

Optically detected thermal stability and biological activity of phage T7 has been compared as the function of the ionic composition and strength of the buffers. The ionic strength range was studied between 20-140 mmol/1. In Tris buffer containing only monovalent ions the biological activity of the phages decreases abruptly below 50 mmol/1 ionic strength. Structural studies show a logarithmic dependence between the ionic strength and the intraphage DNA stability and no significant change in the thermal stability of the whole phage. Mg2+ and Ca2+ ions at low concentration (1 mmol/1) given into a Tris buffer of 20 mmol/1 original ionic strength highly stabilize the biological activity, which stabilization is also to be seen in the intraphage DNA and also in the whole phage thermal denaturation process.

UV optical absorption and circular dichroism (CD) properties (spectra and melting curves) of T7 bacteriophage were investigated to detect "in situ" structural damages which can be related to the biological inactivation due to UV irradiation. UV doses (0.2-1.2 kJ/m2 at 254 nm) near to the biologically effective minimal dose were applied where the initial genetic damage (approximately 10 events/phage) was observed. The decrease of the melting temperature of the helix-coil transition and the broadening of the transition range indicate the destabilization of the intraphage structure due to the presence of about 0.1-0.6% damaged base concentration.

The thermal denaturation as a measure of the structural stability of the nucleoprotein in bacteriophage T7 has been studied in dependence of the ionic environment. Optical density and circular dichroism melting curves measured at wavelengths characterizing either the DNA or protein conformational changes were compared to identify different steps of the denaturation and to follow the effect of the ions. Monovalent salts strengthen the helical structure of intraphage DNA logarithmically in the way as they do in the case of isolated double-stranded DNA. Mg2+ and Ca2+ at very low concentrations stabilize the DNA helicity. Higher divalent ion concentrations decrease the stability of the double helix because of the repulsive ionic interactions. The high structural sensitivity of DNA in the presence of Mg2+ and Ca2+ in this "in situ" environment can be related to the biological role of these ions.