PHOTOREVERSIBLE UV-INACTIVATION OF MESSENGER RNA IN AN INSECT EMBRYO (SMITTIA SPEC., CHIRONOMIDAE, DIPTERA)

Smittia embryos were UV-irradiated during intravitelline cleavage. At this stage, nuclei are heavily shielded by yolk-rich cytoplasm, and do not synthesize detectable amounts of RNA. Irradiation at 265, 285 and 295 nm wavelength caused biological inactivation, and pyrimidine dimer formation in maternal RNA as described earlier (Kalthoff, 1976; Jäckie and Kalthoff, 1978). In addition, we observed marked effects on protein synthesis: (1) The overall rate of [³⁵S]-methionine incorporation in vivo was reduced to less than half of the normal rate. (2) Two-dimensional gel electrophoresis revealed quantitative variations in the synthetic rate of some polypeptides, and the appearance of new ones in UV-irradiated embryos. (3) Translation of polyadenylated RNA from Smittia embryos in a cell-free system was inhibited by UV irradiation in vivo. (4) The apparent degradation, during early embryogenesis, of maternal polyadenylated RNA was retarded in UV-irradiated embryos. Exposure to light (400 nm) after UV caused partial photoreversal of all UV effects observed. Both the photoreactivable sector of UV-inactivation, and the photoreactivated portion of UV inhibition of protein synthesis, were correlated with the amounts of pyrimidine dimers generated in maternal RNA by UV irradiation at the three wavelengths used. These correlated effects were produced most efficiently by 295 nm radiation, indicating the involvement of photosensitizing components in the embryos. Our data show, for the first time to our knowledge, that animal mRNA, after UV irradiation, can be photoreactivated in vivo. Moreover, our results strongly suggest that the photorepairable lesions consist of pyrimidine dimers generated in a photosensitized reaction.     

Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) I. Photosensitized production and light-dependent disappearance of pyrimidine dimers

Abstract. Irradiation of Smittia eggs with UV during intravitelline cleavage causes the formation of pyrimidine dimers in the (largely ribosomal) RNA of the eggs. The yield of dimers is wavelength-dependent in a way that strongly suggests the involvement of photosensitizing egg components. Illumination of UV-irradiated eggs with light (380 or 400 nm) causes both photoreactivation of the eggs and mono-merization of the pyrimidine dimers in their RNA. The photoreactivable sector of the biological damage is correlated with the amount of pyrimidine dimers present in the RNA after inactivation of the eggs with UV of different wavelengths. The data are regarded as the first direct evidence that the photoreactivation of a eukaryotic organism is correlated with the light-dependent (and apparently enzymatic) monomerization of pyrimidine dimers in RNA.     

Photosensitized formation of RNA-protein crosslinks in an insect egg (Smittia spec., Chironomidae, Diptera)

Abstract—Upon irradiation of Smittia eggs with ultraviolet light (UV), the extractability of RNA with phenol decreased. The strongest decrease was observed after irradiation at 295 nm wavelength. After trypsin treatment, RNA could be recovered to the same degree as from unirradiated eggs. The extractability of RNA from irradiated eggs was not enhanced by formamide. dimethyl sulfoxide. high salt concentration or heat treatment. The results suggest that the UV-mediated formation of RNA-protein crosslinks in Smittia eggs involves the action of a photosensitizer.     

Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) II. Evidence for heterogeneous light-dependent repair activities

Abstract. Two biological effects of UV radiation upon Smittia eggs are observed, both of which seem to be associated with the formation of pyrimidine dimers in the RNA (largely ribosomal) of the eggs. While irradiation of the anterior pole region causes the formation of an aberrant segment pattern (double abdomen induction), irradiation of entire eggs leads to an arrest of their development (inactiva-tion). Both UV effects are photoreversible with different action spectra of the photoreactivating light. A dose rate dependence of the photoreactivation can be observed after both UV effects. The saturating dose rate is about 6 W/m² (at 440 nm) after UV induction of double abdomens. Upon UV inactivation, the saturating dose rate level for the photoreactivating light is much higher, and a single light flash causes both a considerable biological reactivation and the disappearance of about 7 × 10⁹ pyrimidine dimers from the total RNA per egg. The results indicate the presence of heterogeneous light-dependent repair activities acting upon UV induced pyrimidine dimers in the RNA of the eggs.     

MULTIPLE EFFECTS OF UV RADIATION (265–330 NM) ON FUNGAL SPORE EMERGENCE

We have investigated the influence of narrow-band UV radiation, 265–330 nm. on germination of spores of the fungus Cladosporium cucumerinum Ellis and Arth., using a Xe arc lamp and filters. Reciprocity of time and dose rate was demonstrated when fungal spores were subjected to UV radiation at 325 nm but failed to hold at 265 nm. Based on these findings, data on fluence response, and partial action spectra, we propose that there are two biologically active sites in this organism that are affected by radiation between 265 and 330 nm and that might be influenced by changes in the stratospheric ozone layer: a short-wave-sensitive site (265–295 nm) and a long-wave-sensitive site (300–330 nm). Data obtained with narrow-band interference filters confirmed previous reports of damage to nucleic acid from UV at 265–295 nm and in addition demonstrated significant inhibition by UV at 300–320 nm. Further studies of the 300 330 nm portion of the spectrum, using combinations of plastic and glass filters, showed that the influence of UV radiation in this region was primarily to produce a non-photoreactivable delay in germ-tube emergence.     

ACTION SPECTRUM FOR PHOTOREACTIVATION OF ULTRAVIOLET-INDUCED MORPHOLOGICAL ABNORMALITY IN SEA URCHIN EGGS

Abstract An action spectrum was obtained for photoreactivation (PR) of morphological abnormality arising from ultraviolet (UV)-irradiation of sea urchin sperm. The wavelength dependence of PR was measured by the restoration of the formation of normal pluteus larvae after the exposure of fertilized eggs to various fluences of monochromatic PR light (313 to 500 nm). The PR action spectrum showed a maximum around 365 nm and a secondary peak somewhere above 400 nm. High PR activity beyond 400 nm wavelengths may reflect an advantageous or adaptational ability to cope with harmful effects of solar UV radiation.     

ACTION SPECTRA FOR UV INDUCTION AND PHOTOREVERSAL OF A SWITCH IN THE DEVELOPMENTAL PROGRAM OF THE EGG OF AN INSECT (SMITTIA)*

Abstract— An action spectrum was established for the induction by ultraviolet (UV) radiation of the aberrant body segment pattern 'double abdomen' in the egg of the Chironomid midge Smittia. The action spectrum shows a peak at 285 nm wavelength, a shoulder at 265 nm, a slight increase from 245 to 240 nm, and a steep decline towards 300 nm. Corrections for wavelength-dependent shielding within the egg result in transformation of the shoulder at 265 nm into a minor peak. These results are compatible with the assumption that a nucleic acid-protein complex is involved in the initial photoreaction. This assumption is supported by the fact that the UV induction of the aberrant body segment pattern 'double abdomen' in the egg of Smittia is photoreversible. Wavelengths effective in this photoreversal range from 310 to 460 nm, with a peak at 440 nm. These spectral characteristics agree with action spectra for photoreactivation in other systems. Indirect photoreactivation does not occur in the egg of Smittia under the conditions of the experiments. Photoreversal with 440 nm radiation at high dose rate is temperature-dependent. The results support the assumption that the molecular basis for photoreversal in the egg of Smittia is similar to the predominant molecular mechanism of direct photoreactivation in other systems. Targets possibly involved in the UV induction of the 'double abdomen' are discussed.     

Correlation of photoreactivation of ultraviolet-induced killing with dimer splitting before and after DNA replication in an excisionless strain of Escherichia coli

Abstract— Splitting of thymine-containing dimers was compared quantitatively with photoreactivation (PR) of killing induced by ultraviolet radiation (254 nm) in a uvrA (excisionless) strain of E. coli. Immediately after irradiation, the splitting rate (number of dimers split/genome/unit PR dose) agreed well with the PR rate of the cells (rate of recovery from photoreactivable lethal damage converted into an ‘estimated’ number of dimers split/genome/unit PR dose). After 4 h of incubation of cells in nutrient medium, the maximal fraction of splittable dimers decreased, as did the maximal fraction of photoreactivable lethal damage. However, the initial splitting rate after incubation was equal to that before incubation. During the 4-h incubation, the heavily irradiated uvrA cells did not divide but became filamentous and their DNA increased about 70 per cent. It is concluded that roughly half of the dimers in DNA that has replicated after ultraviolet irradiation are split as efficiently as those in DNA that has not replicated.     

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.     

PHOTOREACTIVATION OF ICR 2A FROG CELLS AFTER EXPOSURE TO MONOCHROMATIC ULTRAVIOLET RADIATION IN THE 252–313 nm RANGE

Abstract— Exposure of ICR 2A frog cells to photoreactivating light after treatment with monochromatic ultraviolet (UV) radiation in the 252–313 nm range resulted in an increase in survival with similar photoreactivable sectors for each of the wavelengths tested. As photoreactivating enzyme is specific for the repair of pyrimidine dimers in DNA, these findings support the hypothesis that these are critical lesions responsible for killing of cells exposed to UV radiation in this wavelength range. The action spectra for cell killing and production of UV-endonuclease sensitive sites were similar to the DNA absorption spectrum though not identical. Because the number of endonuclease sensitive sites is a reflection of the yield of pyrimidine dimers, these data also suggest that the induction of dimers in DNA by UV radiation in the 252–313 nm range is the principal event leading to cell death.     

ACTION SPECTRUM FOR THE OXYGEN-INDEPENDENT INACTIVATION OF HAEMOPHILUS INFLUENZAE TRANSFORMING DNA WITH NEAR ULTRA-VIOLET LIGHT*

Abstract— The oxygen-independent inactivation of Haemophilis influenzae transforming DNA by near UV light (300–380 nm) has an action spectrum in which the efficiency of inactivation drops rapidly between 313 and 334 nm and more slowly between 334 and 405 nm, with a shoulder between 334 and 365 nm.     

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 WAVELENGTH DEPENDENCE OF HERPES SIMPLEX VIRUS INACTIVATION BY ULTRAVIOLET RADIATION

The effect of different wavelengths of ultraviolet (UV) radiation on Herpes simplex virus when assayed on mammalian cells (measured by plaque forming ability) was investigated. The wavelength dependence of viral inactivation was obtained for 11 different wavelengths over the region 238–297 nm. The resulting action spectrum does not closely follow the absorption spectrum of either nucleic acid or protein. The most effective wavelengths for viral inactivation are over the region 260–280 nm.     

NEAR ULTRAVIOLET INACTIVATION STUDIES ONESCHERICHIA COLI TRYPTOPHANASE AND TRYPTOPHAN SYNTHETASE

Abstract— –The response of two pyridoxal-phosphate-requiring enzymes of E. coli, tryptophanase and tryptophan synthetase, to near UV light (320–400 nm) has been studied. Tryptophanase is inactivated both in vivo and in vitro, but tryptophan synthetase is resistant to near UV under both conditions. This shows that near UV inactivation is not general for pyridoxal-phosphate-requiring enzymes. Substrate protection against light inactivation is demonstrated for tryptophanase. It is furthermore shown that pyridoxal phosphate is required for inactivation of this enzyme. However, the action spectrum for this inactivation does not coincide with the absorption spectrum of tryptophanase or of pyridoxal phosphate.     

EVIDENCE AGAINST DNA AS THE TARGET FOR 334 NM-INDUCED GROWTH DELAY IN ESCHERICHIA COLI*

Abstract— Growth delay was induced with near-UV (334 nm) radiation in Escherichia coli K12 bacterial strains followed by attempts at photoreactivation (PR) of this effect at 405 nm. In the UV-sensitive strain AB2480, a small PR of the observed population growth delay occurred after 334 nm irradiation at 35°C and a much larger PR after 334 nm irradiation at 5°C. However, much of the population growth delay in this strain can be explained as being due to killing, and all or most of the observed PR pertains only to this killed fraction of the population. The true cell growth delay (i.e. that of surviving cells) thus appears to be only slightly, if at all, photoreactivable. This conclusion is supported by studies with a wild-type strain KW8, which shows growth delay at non-lethal doses; this growth delay shows no PR, regardless of the temperature during 334 nm irradiation. These findings indicate that photoreactivable lesions (cyclo-butyl pyrimidine dimers) are not an important cause of near-UV-induced growth delay. Strain AB2480 lacks known dark-repair systems for DNA damage induced by far-UV (below 300 nm) radiation, yet shows the same efficiency for 334-nm-induced growth delay as the wild type, which possesses these dark repair systems. This indicates that lesions in DNA that are dark-repairable by the systems not tunctional in AB2480are not responsible for 334-nm-induced growth delay. It is possible, however, that fragmentary repair systems in AB2480 can operate on some DNA lesion that might cause growth delay. Spontaneously decaying lesions are unlikely, since growth-delay damage decays at a very low rate in non-nutrient medium. Since most of the known types of DNA damage and repair are thus eliminated, these considerations suggest that DNA damage is not involved in near-UV-induced growth delay.     

Photoreactivation of killing in Streptomyces: action spectra and kinetic studies

Abstract— An action spectrum was obtained for photoreactivation of killing (PR) of Streptomyces griseus conidia. This spectrum shows a major peak around 436 nm, originally observed by A. Kelner, and a secondary peak at 313 nm not previously reported. The rate of PR shows a strong dependence upon temperature and dose rate of the PR light at 436 nm, but this decreases to only a slight dependence upon these parameters at 313 nm. These findings suggested that PR at 436 nm in this organism is of the usual photoenzymatic type, but that PR at 313 nm might be of a different kind. A mutant (PHR-1) of S. griseus was found that shows only a narrow range of PR (roughly 310–400 nm) with a single peak at 313 nm. The PR efficiency was lower than for wild type and the PR sector not greater than one-half that of wild type. This PR shows no temperature dependence. Essentially similar behavior was observed with wild-type Streptomyces coelicolor. These findings show that at least some of the PR at 313 nm is a separable phenomenon. It is therefore unlikely to involve a mechanism identical to that at 436 nm. The nature of PR at 313 nm in Streptomyces is not known. If it is enzymatic, it is remarkable in having little or no dependence upon temperature and dose rate. Absence of photoprotection and liquid-holding recovery indicate that it is not indirect PR. Some of it (that part exhibited by S. griseus PHR-1 and S. coelicolor) might result from a direct photochemical action on DNA.     

Experimental Correspondence between Spore Dosimetry and Spectral Photometry of Solar Ultraviolet Radiation

Abstract— The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelength interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (±SD) between them was 1.24 (±0.16) for 14 data points showing high inactivation rates (<0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.     

Ultraviolet Spectral Energy Differences Affect the Ability of Sunscreen Lotions to Prevent Ultraviolet-Radiation-Induced Immunosuppression*

Acute exposure to UV radiation causes immunosuppression of contact hypersensitivity (CH) responses. Past studies conducted with unfiltered sunlamps emitting nonsolar spectrum UV power (wavelengths below 295 nm) or using excessive UV doses have suggested sunscreens may not prevent UV-induced immunosuppression in mice. This study was thus designed to evaluate critically the effects of different UV energy spectra on the immune protection capacity of sunscreen lotions. Minimum immune suppression doses (MISD), i.e. the lowest UV dose to cause~50% suppression of the CH response to dinitrofluorobenzene in C3H mice, were established for three artificial UV sources. The MISD for each UV source was 0.25 kJ/m² for unfiltered FS20 sunlamps (FS), 0.90 kJ/m² for Kodacel-filtered FS20 sunlamps (KFS), which do not emit UV power at wavelengths <290 nm, and 1.35 kJ/m² for a 1000 W filtered xenon arc lamp solar simulator. Using MISD as baseline, sunscreens with labeled sun protection factors (SPF) of 4, 8, 15 and 30 were tested with each UV source to establish their relative immune protection factors. The immune protection factor of each sunscreen exceeded its labeled SPF in tests conducted with the solar simulator, which has a UV power spectrum (295–400 nm) similar to that of sunlight. Conversely, sunscreen immune protection factors were significantly less than the labeled SPF in tests conducted with FS and KFS. Comparison of the immunosuppression effectiveness spectra showed that relatively small amounts of nonsolar spectrum UV energy, i.e. UVC (200–290 nm) and/or shorter wavelength UVB (between 290 and 295 nm), produced by FS and KFS contributes significantly to the induction of immunosuppression. For example, 36.3% and 3.5% of the total immunosuppressive UV energy from FS and KFS, respectively, lies below 295 nm. Sunscreen absorption spectra showed that transmission of immunosuppressive UV energy below 295 nm for FS was at least eight-fold higher than that for KFS. Compared to the solar simulator UV spectrum the transmission of nonsolar immunosuppressive UV energy through sunscreens was >15-fold higher for FS and ≥1.5-fold higher for KFS. These data demonstrate that relevant evaluations of sunscreen immune protection can only be obtained when tests are conducted with UV sources that produce UV power spectra similar to that of sunlight and UV doses are employed that are based on established MISD.     

THE WAVELENGTH DEPENDENCE OF 8-METHOXYPSORALEN PHOTOSENSITIZATION OF HOST CAPACITY INACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

Abstract— The addition of 8-methoxypsoralen to cultures of African green monkey cells (CV-I) sensitized the inactivation by near UV radiation (302–370 nm) of the ability of the cells to host herpes simplex virus. No sensitizing effect by drug addition was noted for far UV radiation (232–297 nm). An action spectrum for the photosensitized inactivation of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum of 8-methoxypsoralen.     

Oxygen-independent inactivation of Haemophilus influenzae transforming DNA by monochromatic radiation: action spectrum, effect of histitine and repair.

Abstract— The action spectrum for the oxygen-independent inactivation of native transforming DNA from Haemophilus influenzae with near-UV radiation revealed a shoulder beginning at 334 and extending to 460 nm. The presence of 0.2 M histidine during irradiation produced a small increase in inactivation at 254, 290 and 313 nm, a large increase at 334 nm and a decrease in inactivation at 365, 405 and 460 nm. Photoreactivation did not reverse the DNA damage produced at pH 7.0 at 334, 365, 405 and 460 nm, but did reactivate the DNA after irradiation at 254, 290 and 313 nm. The inactivation of DNA irradiated at 254, 290 and 313 nm was considerably greater when the transforming ability was assayed in an excision-defective mutant compared with the wild type, although DNA irradiated at 334, 365, 405 and 460 nm showed smaller differences. These results suggest that the oxygen-independent inactivation of H. influenzae DNA at pH 7 by irradiation at 334, 365, 405 and 460 nm is caused by lesions other than pyrimidine dimers.