INACTIVATION OF A RHIZOBIUM BACTERIO-PHAGE BY ULTRAVIOLET RADIATION OF DIFFERENT WAVE-LENGTHS

Abstract— The action spectrum for inactivation of a Rhizobiurn bacteriophage by U.V. radiation follows the shape of the absorption spectrum of DNA between the wave-lengths of 240 and 290 mμ (where inactivation probably reflects damage to the nucleic acid only), and deviates sharply upwards at wave-lengths shorter than 240 mμ (where inactivation may depend on damage to both the nucleic acid, and protein of the phage). The rate of inactivation follows first order kinetics approximately at all wave-lengths tested. Infectivity of the phage is halved when each mg of the phage nucleic acid has absorbed about 0 08 J of radiation energy at any wave-length between 240 and 290 mμ.
The bacteriopharge can be photoreactivated after inactivation at any wave-length between 230 and 290 mμ, but less so after inactivation at 230 mμ than at any wave-length above 240 mμ. No evidence was found to suggest that dimerization of thymine residues of the phage-DNA plays any part in the mechanism of inactivation of the bacteriophage by U.V. radiation.     

ACTION SPECTRUM FOR INACTIVATION OF THE INFECTIVITY OF POTATO VIRUS X BY U.V. RADIATION

Abstract— Quantum yields for inactivation of infectivity of potato virus X by monochromatic ultraviolet radiation of wavelengths ranging from 230 to 290 nm, were measured with reference to energy absorbed by (a) the whole virus and (b) the virus RNA. The yields depended on the wavelength, but those with reference to energy absorbed by the RNA varied much less (with extreme values of 10^-3 and 1.9 ± 10^-3 than those with reference to whole virus. Consequently the action spectrum for inactivation of a dilute solution of the virus resembled the shape of the absorption spectrum of the RNA, but not closely enough to allow coincidence by adjusting the scales. The amount of photoreactivation increased as the wavelength increased and also as the year progressed from May to July; the extreme values of the photoreactivable sector were 0.43 and 0.86.     

THE INACTIVATION OF RIBONUCLEIC ACID FROM TOBACCO MOSAIC VIRUS BY ULTRAVIOLET RADIATION AT DIFFERENT WAVE-LENGTHS

Abstract— Although both thymine and uracil can form similar dimers, exposing RNA of tobacco mosaic virus lo ultraviolet radiation of different wavelengths did not reproduce any of the phenomena that implicate dimerization of thymine residues as a major cause of the inactivation of a bacterial transforming DNA. If uracil residues dimerize at all in the irradiated RNA, such dimerization either does not affect infectivity or is not photoreversible in the same way as dirnerization of thymine residues in DNA. Unlike inactivation of the transforming DNA, inactivation of the virus-RNA seems to be a function of the amount of absorbed radiation energy, irrespective of the wave-length within the range 285 to 230 mμ and irrespective of a change in the wave-length during irradiation.     

ULTRAVIOLET IRRADIATION OF POTATO VIRUS X, ITS RNA, AND A HYBRID VIRUS PARTICLE: PHOTOREACTIVATION, KINETIC ISOTOPE EFFECTS, AND QUANTUM YIELD OF INACTIVATION*

Abstract— Studies have been made of the inactivation of potato virus X (PVX), free PVX-RNA, and a phenotypically mixed virus particle composed of PVX-RNA and tobacco mosiac virus (U-1 strain) protein (PVX_RNA: TMV_PRO) by ultraviolet radiation of 254, 280 and 302 nm wavelengths. Rate constants and quantum yields of inactivation have been determined under conditions of photoreactivation and non-photoreactivation in H₂O and D₂O. The ratios of the rate constants in H₂O to those in D₂O obtained for PVX-RNA were greater than unity at the above three wavelengths, as was the case for PVX at 254 and 302 nm. The ratios of the rate constants were, within experimental error, equal to unity for PVX_RNA:TMV_PRO at all three wavelengths and for PVX at 280 nm. It is concluded that, in contrast to the situation observed with TMV and PVX_RNA:TMV_PRO, the behavior of the intact PVX viron upon irradiation closely approximates the behavior of free RNA in solution, on the basis of quantum yields of inactivation, photoreactivated sector, and the ratio of kinetic isotope effects in H₂O and D₂O.     

MECHANISM OF PHOTOINACTIVATION OF PLANT PLASMA MEMBRANE ATPASE

Abstract UV radiation at 290 and 365 nm inactivates two forms of the K⁺-stimulated ATPase associated with the plasma membrane of suspension-cultured cells of Rosa damascena . One form is 15 and 36 times more sensitive than the other to 290 and 365 nm, respectively. For both forms, the inactivation requires oxygen, is inhibited by azide and diazobicyclo(2.2.2.2)octane, but not glycerol, and is enhanced up to 7.5 times in deuterium oxide solvent. Inactivation occurs concomitantly with loss of absorbance at 290 nm. Cs⁺ and NO⁻₃, quenchers of tryptophan fluorescence, inhibit inactivation. The results suggest that inactivation involves singlet-oxygen mediated destruction of tryptophans in the ATPases.     

INFLUENCE OF pH AND OXYGEN ON 315 mμ INACTIVATION AND THYMINE DIMERIZATION IN MUTANTS OF BACTERIOPHAGE T4

Abstract— 1. Irradiation with 315 mμ light inactivates phage T4v-x C, and T4v^-x- , and forms thymine dimers in their DNA.
2. Both the rates of inactivation and of thymine dimerization depend upon pH and gaseous environment during irradiation. The U.V. sensitivities are: 1 (pH 7, N₂, 03, 2.2 (pH 3.5, Oz), 3.3 (pH 3–5, N₂; and the corresponding rates of thymine dimerization 1: 2.5: 5.2. The number of thymine dimers per lethal hit observed withT4v-x + are: 5.7 (pH 7, N₂, O₂, 5.4 (pH 3.5, O₂, 10.9 (pH 3.5, N₂); and forT4v-x-: 4.6, 3.4, and 7.1 with the same sequence of conditions.
3. Also the photoreactivable sectors depend upon the environmental conditions at 315 mp inactivation. In T4v-x f this sector amounts to about 50 per cent at pH 7, 18 per cent at pH 3.5, O., and 29 per cent at pH 3.5, N, respectively.
4. The molecular basis of these findings is discussed. It is concluded that, besides thymine dimer, at least one other lethal photoproduct (probably a photoproduct of cytosine) is involved in photoreactivation.     

INDUCTION OF DIRECT AND INDIRECT SINGLE-STRAND BREAKS IN HUMAN CELL DNA BY FAR- AND NEAR-ULTRAVIOLET RADIATIONS: ACTION SPECTRUM AND MECHANISMS

Abstract— An action spectrum for the immediate induction in DNA of single-strand breaks (SSBs, frank breaks plus alkali-labile sites) in human P3 teratoma cells in culture by monochromatic 254-, 270-, 290-, 313-, 334-, 365-, and 405-nm radiation is described. The cells were held at +0.5C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D₂O environment (compared with the normal H₂O environment) did not alter the rate of induction of SSBs by 290-nm radiation, whereas the D₂O environment enhanced the induction of SSBs by 365- and 405-nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs . wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far-UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near-UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365-nm radiation.     

DIFFERENT (DIRECT and INDIRECT) MECHANISMS FOR THE INDUCTION OF DNA-PROTEIN CROSSLINKS IN HUMAN CELLS BY FAR- and NEAR-ULTRAVIOLET RADIATIONS (290 and 405 nm)

Abstract— Apparent DNA-protein crosslinking induced by monochromatic 290 and 405 nm Tadiations was measured in cultured human P3 teratocarcinoma cells with DNA alkaline elution techniques. The rates of the induction of crosslinks by 290 nm radiation were the same when the cells were irradiated either aerobically or anaerobically or when the cells were in an H₂O or D₂O aqueous environment. With 405 nm radiation, anaerobic irradiation reduced the induction of the crosslinks (dose modifying factor is about 0.2), and about twice as many crosslinks were observed when the cells were irradiated in an environment of D₂O rather than H₂O. The results are consistent with the hypothesis that far-UV radiation induces DNA-protein crosslinks by a direct mechanism, whereas near-UV radiation induces crosslinks via indirect photodynamic photosensitizations in which unidentified cellular endogenous photosensitizers and reactive species of oxygen are used.     

ANTIVIRAL ACTIVITY OF GILVOCARCIN V PLUS UVA RADIATION

Abstract Gilvocarcin V (GV), a coumarin, is a nucleic acid photosensitizer that is phototoxic to bacteria and mammalian cells at picomolar levels in the presence of near-UV radiation (UVA). We evaluated the effectiveness of GV plus UVA for inactivation of several viruses, including herpes simplex virus, type 1 (HSV) and the bacterial viruses φX174, T7, PRD1 and φ6. Some inactivation of the bacterial viruses was observed with UVA radiation alone (4–50% survival at 26 kJ/m²). Additional photosensitized inactivation was observed only with T7 and φ6 at 2.0 μ M GV. On the other hand, HSV was photoinactivated with concentrations of GV three orders of magnitude lower (1.0 n M ). Similar to the case with UV (254 nm) inactivation, the GV-UVA survival curve for HSV indicated multicomponent inactivation kinetics, which could not be explained by photobleaching of GV. The wide range of photosensitivities of these viruses to GV cannot be adequately explained by models based only on viral nucleic acid content or presence of lipid envelopes.     

STUDIES OF THE ROLE OF THE COAT PROTEIN IN THE U.V. PHOTOINACTIVATION OF U(1) AND U(2) STRAINS OF TMV*

Abstract— Two properties of the u.v. inactivation process in the u.v. sensitive U(2) strain have been investigated: (1) The increased binding of protein to RNA induced by irradiation of the virus at 254 nm; (2) The action spectrum for u.v. inactivation of U(2) between 250 nm and 285 nm. The extent of the u.v. induced binding of protein to RNA is similar to that previously found in the resistant U(1) strain, thereby eliminating the possibility that the capacity for this binding phenomenon bears any correlation to the difference in u.v. sensitivities of these two viruses at 254 nm. The results indicate that the radiation induced interaction of protein and RNA in U(1) and U(2) are probably similar. The action spectrum for U(2) resembles the absorption spectrum of the RNA between 250 nm and 285 nm implicating the RNA as the primary absorber leading to inactivation of the virus in this region of the spectrum. Quantum yields calculated for U(2) virus and free TMV-RNA irradiated at 254 nm reveal that the irradiated free RNA may be as much as 1–4 times more sensitive to inactivation at this wavelength than RNA in the intact virus. It is concluded that the coat protein of U(2) probably offers some protection to the enclosed RNA against u.v. damage at 254 nm, therefore, the difference in u.v. sensitivity between U(1) and U(2) TMV at this wavelength is a consequence of a difference in the degree of protection offered by the respective coat proteins to the enclosed RNA.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

PHOTOSENSITIZED INACTIVATION OF DNA BY MONOCHROMATIC 334-nm RADIATION IN THE PRESENCE OF 2-THIOURACIL: GENETIC ACTIVITY AND BACKBONE BREAKS

Abstract Monochromatic 334-nm radiation delivered under aerobic conditions inactivates the genetic activity (ability to transform auxotrophic recipient cells to nutritional prototrophy) of isolated transforming Bacillus subtilis DNA. The presence of superoxide dismutase (SOD), catalase, and mannitol reduces the 334-nm inactivation. The rate of inactivation of the genetic activity by 334-nm radiation is enhanced fivefold by the sensitizer 2-thiouracil (s²Ura). This enhancement is substantially reversed when the irradiations are performed in the presence of mannitol, and, to a lesser extent, SOD. Catalase slightly reduces the s²Ura enhancement of 334-nm inactivation of transforming activity. Backbone breaks induced in the same DNA by aerobic 334-nm radiation were also enhanced markedly by the presence of s²Ura; this enhancement was reversed by the presence of mannitol and, to a lesser extent, SOD during irradiation. Catalase had no effect upon s²Ura-enhanced, 334-nm-induced SSBs. Whereas DNA breakage may be responsible for a portion of the inactivation of the DNA by the photosensitized reaction between s²-Ura and 334-nm radiation, it is not the only inactivating lesion, because the yield of SSBs per lethal hit per unit length of DNA is not constant for all the irradiation conditions studied. The results support a complex role for active oxygen species in inactivation of transforming activity and DNA breakage by s²Ura-enhanced 334-nm radiation. They are also consistent with the formation of superoxide anion, hydroxyl radical, and possibly also singlet molecular oxygen, generated from ground-state molecular oxygen by reactive s²Ura in both Type I and II reactions.     

RNA—PROTEIN CROSSLINK AND RNA CHAIN BREAK FORMATION ON UV-IRRADIATION OF TOBACCO MOSAIC VIRUS

Abstract— The ability of UV-irradiation (254 nm) to induce formation of RNA-protein crosslinks in tobacco mosaic virus (TMV) particles have been studied by Cs₂SO₄ density gradient centrifugation, analytical centrifugation, nitrocellulose filter binding and two-dimensional peptide mapping. RNA-protein crosslinks were found to be formed on UV-irradiation of TMV, but the parallel process of UV-induced RNA chain breakage complicated their quantitation. Using speciall devised equations, the quantum yield of RNA-protein crosslink formation was found to be 0.65 × 10⁻⁵ and that of RNA chain break formation 0.95 × 10⁻⁵.     

FLUORESCENCE OF THYMINE IN AQUEOUS SOLUTION AT 300° K

Abstract— –Fluorescence of thymine in neutral aqueous solution at room temperature has been detected using the multiscaling operation of a multichannel analyzer. The emission maximum (2.96 μm^-1) and 0-0 transition energy (3.37-3.45 μm^-1) are close to those determined at liquid nitrogen temperature in mixed solvents. The quantum efficiency of fluorescence excited at 3.77 μm^-1 is calculated to be 1.04 × 10^-4.
The corrected relative excitation spectrum shows significant differences from the absorption spectrum when both are determined under identical conditions of concentration and spectral bandwidth on the same instrument. The quantum yield of fluorescence decreases about 2-fold as the energy of excitation is increased beyond the 0-0' transition and follows the relation 1/φ°α E _excit..
This behavior is discussed in terms of (a) n π* and ππ* states, (b) emission from a minor tautomer and (c) kinetics of competing deactivation processes.     

APPLICATION OF PHOTODYNAMIC OXIDATION TO THE DISINFECTION OF TAPWATER, SEA WATER, AND SEWAGE CONTAMINATED WITH POLIOVIRUS

Abstract. Poliovirus when added to tapwater, sewage or seawater was readily photoinactivated by methylene blue and visible light. Typically, almost 2.5 logs of virus could be inactivated upon a 5-min exposure to 670 nm light (20 W/m²) in solutions containing 13 μ M methylene blue at pH 10.0. A biphasic inactivation curve was produced for poliovirus, regardless of dye concentration, pH, temperature, sensitization time, nature of suspending solution or sequence of light exposure. These results indicated that a multi-hit inactivation event was occurring. Preincubation of the dye-virus mixture at 24°C increased the rate of virus photoinactivation. Dye concentrations above 26 μ M have little advantage in increasing the amount of virus photoinactivated. Significant inactivation of the virus in the dark occurred at high dye concentrations (52–130μ M ).     

FORMATION OF THYMINE DIMERS IN MAMMALIAN SKIN BY ULTRAVIOLET RADIATION IN VIVO

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [³H]-labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn-producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [³H]-thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn-producing spectrum (290–320 nm), may be related to the formation of such dimers.     

INACTIVATION BY ULTRAVIOLET RADIATION AT DIFFERENT WAVELENGTHS OF THE RNA ISOLATED FROM POTATO VIRUS X

Abstract— –The action spectra and quantum yields for photoreactivable, non-photoreactivable and total damage caused by u.v. in the RNA isolated from potato virus X differ from those for similar types of damage in the whole virus. The differences result from the virus protein partly protecting the RNA from damage, and the degree of protection depends on the wavelength of u.v. and on the salt concentration of the irradiated solution. The action spectra for the different types of damage in the RNA all resemble the absorption spectrum of the RNA, but do not exactly parallel it. The photoreactivable sectors of the RNA and of the whole virus are greater at 290 nm than at 230 nm but, whereas that of the virus increases rectilinearly, that of the RNA has a pronounced minimum at about 250 nm. At wavelengths longer than 240 nm, the photoreactivable sector of the virus exceeds that of the RNA, because, at these wavelengths, the virus protein protects the RNA more against non-photoreactivable damage than against photoreactivable damage.     

THE ELECTROPHOTOLUMINESCENCE OF CHLOROPLASTS

Abstract— Delayed light emission emanating from preilluminated chloroplasts can be perturbed with pulsed DC electric fields (200–4000 V cm^-1), The perturbation produces a strong stimulation of chlorophyll luminescence. During the field perturbation the stimulated emission rises to a maximum, typically within 100μs. and then decays. Two kinetic components, R (rapid) and S (slow)†, are distinguished on the basis of their rise and decay times and their field-dependence. The R component increases exponentially at high fields, decays within 100–300μs during the field pulse and collapses with t _1/2= 15 μs at the end of the field pulse. The S component occurs at low fields, exhibits near saturation at 500 V cm^-1, decays with t _1/2 about 3 ms during the field pulse, and collapses with t _1/2= 38μs at the end of the field pulse. Studies using inhibitors, ionophores, electron donors and electron acceptors associate the R component with ion transport processes. The relation to electron transport associated with Photosystem II is discussed.     

OBSERVATIONS ON THE PHOTOSENSITIZED BREAKAGE OF DNA BY 2-THIOURACIL AND 334-nm ULTRAVIOLET RADIATION

Abstract— Breaks induced in purified DNA by 334-nm ultraviolet (UV) radiation are enhanced 30 times when 2-thiouracil (s²Ura) is present during aerobic irradiation. This enhancement by s²Ura is maximally effective at a concentration of about 1 m M. Anoxic irradiation reduces the s²Ura-enhanced breakage by 90%, indicating a Type II photosensitization. Benzoate, glycerol, diazabicyclo[2.2.2.]octane (DABCO) and histidine all inhibit formation of s²Ura photosensitized breaks, unlike diethylenetriaminepenta-acetic acid (DETAPAC) and catalase, which do not. The relationships between the concentration of DABCO. benzoate and histidine and their protection against induction of single strand breaks (SSBs) were similar, with little inhibition below 10 m M and maximal inhibition near 0.1 M for all compounds. Irradiation of the DNA-s²Ura mixture dissolved in D₂O instead of H₂O enhanced the rate of induction of SSBs in DNA by 334-nm light almost five times. Addition of superoxide dismutase (40, 80 and 200 μg/ml) decreased the rate of induction of breaks in DNA by 334-nm radiation plus s²Ura (in H₂O) by about 40%. Boiled superoxide dismutase had no effect.     

MECHANISM OF HIGH POWER PICOSECOND LASER UV INACTIVATION OF VIRUSES AND BACTERIAL PLASMIDS

Abstract— The mechanism of inactivating action of high-power picosecond laser UV radiation (λ= 266 nm) on the λ and φX174 bacteriophages and the pBR 322 plasmid has been studied. It has been shown that at UV radiation intensities from 10¹¹ to 10¹³ W/m², inactivation of viruses and bacterial plasmids occurs mainly on account of single-strand breaks in the DNA chain unlike the case of less powerful UV radiation where the inactivation is associated with the formation of pyrimidine dimers.