EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

REPAIR OF CYCLOBUTANE DIMERS AND (6–4) PHOTOPRODUCTS IN ICR 2A FROG CELLS

Abstract— The removal of cyclobutane dimers and Pyr(6–4)Pyo photoproducts from the DNA of UV-irradiated ICR 2A frog cells was determined by radioimmunoassay. In the absence of photoreactivat-ing light, 15% of the cyclobutane dimers and 60% of the (6–4) photoproducts were removed 24 h post-irradiation with 10 J m⁻², Exposure to 30 kJ m⁻² photoreactivating light resulted in removal of 80% of the cyclobutane dimers and an enhanced rate of repair of (6–4) photoproducts, resulting in a loss of 50% of these lesions in 3 h. The preferential removal of (6–4) photoproducts by excision repair resembles previously published data for mammalian cells.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

PHOTOREVERSAL OF ABNORMAL MORPHOGENESIS IN SEA-URCHIN EMBRYOS CAUSED BY UV-IRRADIATION

Abstract— Morphological abnormalities induced by UV-irradiation of 8- or 16-cell-stage embryos of the sea-urchin, Hemicentrotus pulchenimus , and their photoreversal were studied. UV-irradiation of the animal hemisphere of embryos caused the formation of exogastrulae, while that of the vegetal hemisphere caused the formation of permanent blastulae. These UV-induced morphological abnormalities were photoreversed when the UV-irradiated embryos were subsequently illuminated with visible light, so that the UV-irradiated embryos developed into normal pluteus-larvae. When UV-irradiated embryos were illuminated with visible light up to the onset of the DNA-synthesis phase of the following cell cycle, the UV-induced morphological abnormalities were photoreversed almost completely. The effectiveness of an exposure to visible light declined thereafter and was subsequently completely lost.     

RELATIONSHIP BETWEEN SURVIVAL AND THYMINE-DIMER EXCISION IN ULTRAVIOLET-IRRADIATED ESCHERICHIA COLI

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. light and excision of thymine dimers of bacterial strains E. coli B/r hcr ⁺ thy- trp ^-, E. coli B/r hcr ^-thy^- trp ^-, and E, coli 15 T^- 555–7 thy ^- meth ^- trp ^- arg ^- has been studied.
The prestarvation increased the resistance of all the strains but reasonably inhibited excision of thymine dimers. Thus the enhancement of u.v. resistance after amino acid prestarvation was not due to more complete excision of thymine dimers.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED SISTER CHROMATID EXCHANGES IN POTOROUS CELLS

Abstract— Exposure to visible light after UV-irradiation showed a remarkable effect on UV-induced sister chromatid exchanges (SCEs). After 6-h exposure to visible light (3 × 10⁵ J/m²), two-thirds of the UV-induced SCEs were prevented, confirming Kato's findings. Exposure to visible light before UV irradiation had no effect. This effect of visible light on UV-induced SCEs was temperature dependent, suggesting the presence of enzymatic photoreactivation.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

Replication in UV-lrradiated Caenorhabditis elegans Embryos

Abstract— Replication continues in wild-type (but not rad mutant) Caenorhabditis elegans embryos even after exposure to massive fluences of UV radiation. It is of interest to elucidate the mechanism(s) for this "damage-resistant" DNA synthesis. In this study, DNA from unirradiated and UV-irradiated wild-type embryos was examined using the electron microscope. Large fluences of UV radiation (180 J m⁻²) had little effect on either replication bubble size or distances between bubbles in wild-type embryos, indicating that the damage-resistant DNA synthesis was not grossly aberrant. Conversely, UV irradiation significantly decreased center-to-center distances between bubbles in excision-repair-deficient rad-3 embryos. This suggests that the decreased DNA synthesis observed after UV irradiation in rad-3 embryos is due largely to blockage of elongation of DNA synthesis.     

INTERACTION OF RESTORATION PROCESSES IN UV IRRADIATED ESCHERICHIA COLZ CELLS

Abstract— An attempt was performed to estimate survival and course of DNA synthesis in Escherichia coli B/r hcr' and hcr^- cells in relation to the amount of unexcised dimers.
In exponential growing hcr⁺ cells irradiated with 30 Jm^-2, dimers were almost completely excised and survival of cells was equal to about 3%. In the hcr⁺ cells prestarved for amino acid and thymine and irradiated by the same fluence, survival of cells was almost equal while two thirds of dimers remained unexcised and could be detected in the hybrid DNA consisting of parental and daughter chains. In exponentially growing hcr^+- cells irradiated with 20Jm^-, the same amount of dimers was produced which remained unexcised in the prestarved hcr⁺ cells. However, their survival was equal to about 0.02%.
Despite the great differences in dimer contents, about one third of DNA was replicated after UV in both exponentially growing and prestarved hcr+ cells producing well defined HL-hybrid peak, and the newly synthesized DNA was normal-sized. In hcr⁺ cells which contained approximately the same amount of dimers as in hcr⁺ prestarved cells, the amount of replicated DNA was too low to form a detectable density labelled hybrid peak, and the newly synthesized DNA was in short pieces.
Thus, when hcr⁺ and hcr^+- cells contain the same number of residual dimers, they have different levels of tolerance to these dimers.     

THE EFFECTS OF HIGH-INTENSITY UV-RADIATION ON NUCLEIC ACIDS AND THEIR COMPONENTS—I. THYMINE

Abstract— The set of final products of thymine conversion induced by high-intensity UV irradiation (λ= 266nm, intensity 10²⁴-5 × 10²⁹ photons·s⁻¹·m⁻², pulse duration 10ns) of the dilute aqueous solution to the first approximation is similar to that formed with ionizing irradiation (γ-irradiation of aqueous solution or autoradiolysis of a solid 2-[¹⁴C]-thymine). The data obtained suggest that high-intensity UV-induced photochemical conversion of thymine involves photoionization and/or photodissociation. These processes pass through the higher excited state(s) populated as a result of the second photon absorption by excited (most probably in the T₁ triplet state) thymine molecules.     

THE IN VIVO ASSOCIATION OF MANGANESE WITH THE CHROMOSOME OF MICROCOCCUS RADIODURANS

Abstract —Extremely high levels of paramagnetic manganese (Mn²⁺) which quench phosphorescent reactions have been found to inhibit the formation of thymine-containing dimers in M. radiodurans . Lowering the concentration of Mn²⁺ in the culture medium resulted in a lower intracellular concentration of Mn²⁺, an increase in the UV-sensitivity of this bacterium, and a larger photochemical yield of thymine-containing dimers. High levels of paramagnetic Mn²⁺ were not found in other test organisms which are more sensitive to UV-irradiation. One interpretation of our data is that in Micrococcus radiodurans Mn²⁺ may bind to the chromosome and thereby reduce the photochemical yield of thymine-containing dimers.     

MULTIPLE ACTION OF FAR-RED LIGHT IN PHOTOPERIODIC INDUCTION and CIRCADIAN RHYTHMICITY

Abstract— It is generally accepted that phytochrome influences the photoperiodic induction of flowering through its interaction with the circadian clock mechanism. We have attempted to separate the effects of phytochrome on the clock mechanism from those that mediate flowering directly by examining a number of responses that are unrelated to flowering but are also regulated by the circadian clock. Gas exchange measurements of both CO₂ and H₂0 vapor were monitored under light conditions (200 μmol m ² s⁻¹) where the addition of far-red energy is required for the maximal promotion of flowering. In addition, photosynthetic capacity and maximal transpiration rates were measured in plants grown under continuous dim (20 μmol m⁻² S⁻') light, with or without supplemental far-red, by exposing them briefly to saturating fluxes (1000 μmol m⁻² s^-l) of light. Net CO₂ fixation was very weakly rhythmic in plants grown under both high and low light and this weak oscillation was completely suppressed by far-red light. Far-red also suppressed the rhythm in transpiration under high light, but the rhythm was immediately reinstated when the far-red light was removed. The phase of this rhythm was also reset with the next peak always occurring15–18 h after the far-red was turned off. When grown under dim light, the transpiration rhythm was not suppressed and the amplitude of the oscillation was more than doubled. Far-red light appears to interact with the rhythm in transpiration in a manner suggesting that the stomatal rhythm may be coupled to the same clock oscillator that regulates the flowering rhythm.     

ULTRAVIOLET-INDUCED REACTIONS OF THYMINE AND URACIL IN THE PRESENCE OF CYSTEINE

Abstract —Ultraviolet-radiation photolysis of thymine in the presence of cysteine gives rise to four isomeric dimers, dihydrothymine, and at least five cysteine addition products. Similar reactions occur for uracil but the products have not all been characterized in detail. The addition reactions arise from the triplet state of the pyrimidine. The initial step is production of a hydropyrimidine radical, which then reacts with cysteine to give the addition products. The triplet is quenched by cysteine with a rate constant of about 2 times 10⁸ M^-1 s^-1 for thymine and 2–9 times 10⁸ for uracil. The total yield of products gives a lower-limit estimate of the triplet yield and hence of the intersystem-crossing efficiency. These studies, combined with earlier determinations of dimer yields, show that 93% of the thymine triplets which interact with another thymine molecule are quenched without forming stable dimers. For uracil, the corresponding figure is 75%.     

THYMINE DIMERIZATION IN L-STRAIN MAMMALIAN CELLS AFTER IRRADIATION WITH ULTRAVIOLET LIGHT AND THE SEARCH FOR REPAIR MECHANISMS

Abstract— The formation of thymine dimers in the DNA of L -strain mammalian cells after irradiation with ultraviolet light has been demonstrated. The amount of dimer formed rises with the dose of u.v. light.
In the course of post-irradiation incubation the thymine dimers remain in the TCA insoluble fraction and diminish as did the other thymidine-H³ derivatives with increasing incubation time. The dimer is not found in the soluble fraction. Thus, dimer excision (i.e. its liberation into the soluble fraction) as an expression of repair of radiation damage analogous to dark repair in E. coli was not found in these experiments.     

SEASONAL VARIATION OF SOLAR UV-RADIATION AT A HIGH MOUNTAIN STATION

Abstract— At the high mountain station Jungfraujoch (3576 m), the maximum daily totals for erythemal dose (G_ER), UV-A radiation (G_UVA) and global radiation (G) are 29 Sunburn Units d⁻¹, l.7 MJ m⁻²d⁻¹ and 37 MJ m⁻² d⁻¹. The maximum instantaneous values at solar noon in midsummer are 4.2 Sunburn Unit h⁻¹, 53 W m⁻² and 1110 W m⁻². A significantly nonlinear relation between G_ER and G results from the influence of the irradiated ozone mass on the UV-B erythemal dose. In contrast, G_UVA and G are linearly proportional, which can be seen from the diurnal and seasonal courses of the ratios G_ER/G and G_UVA/G AND from their dependence on the optical air mass. UV-A radiation flux is less attenuated by cloudiness than is global radiation. This effect is masked for the erythemal dose by variations in the ozone concentration. Due to seasonal ozone layer thickness and effective pathlength variations, the ratio G_ER/G shows a significant asymmetry. At the autumn equinox it is about 16% higher than at the spring equinox.     

Porphyrins and Related Compounds as Photoactivatable Insecticides I. Phototoxic Activity of Hematoporphyrin Toward Ceratitis capitata and Bactrocera oleae

The photodynamic sensitizer hematoporphyrin (HP) IX was efficiently accumulated by Ceratitis capitata (Mediterranean fruit fly) and Bactrocera oleae (olive fly) when the insects were fed with a sugar/protein bait containing micromolar amounts of porphyrin. Hematoporphyrin appeared to be mainly accumulated in the midgut, Malpighian tubes, adipose tissue and cuticle and was gradually cleared from the organism in a 24–48 h time interval. Exposure of the HP-fed flies to light mimicking the solar spectrum caused a decrease in the survival whose extent was modulated by the HP concentration in the bait, the irradiation fluence rate and the total light dose. For 8 μmol/mL HP in the bait 100% mortality during 1 h exposure to light was obtained using a fluence rate of 1220 μE s⁻¹ m⁻² for C. capitata and 2080 μE s⁻¹ m⁻² for B. oleae . The latter fly was somewhat less photosensitive than C. capitata possibly owing to the smaller amount of ingested HP and/or darker pigmentation. Studies are in progress in order to extend these investigations from the laboratory to the field scale.     

SENSITIVITIES TO MONOCHROMATIC 254-nm AND 365-nm RADIATION OF CLOSELY RELATED STRAINS OF Saccharomyces cerevisiae WITH DIFFERING REPAIR CAPABILITIES

Abstract— Sensitivity to monochromatic 254- and 365-nm radiation was compared in closely related yeast strains with defects in one or more of the excision-repair ( rad1 ), error-prone repair ( rad18 ), or recombinational-repair ( rad51 ) pathways. At 254 nm, mutants defective in a single repair pathway exhibited slight to moderate UV sensitivity; those defective in two separate pathways were somewhat more UV sensitive, while triple mutants defective in all three pathways exhibited extreme UV sensitivity with a lethal event corresponding to 0.05 J m⁻². Repair defects also rendered mutants sensitive to 365-nm radiation; strains with single defects exhibited slight sensitivity, mutants with two defective pathways were more sensitive, and triple mutants exhibited maximal sensitivity with a lethal event corresponding to 2.4 times 10⁴ J m⁻². In the triple mutant ( rad1, rad18, rad51 ) at both 254 and 365 nm, the dose per lethal event was almost identical with comparable values in a repair-deficient double mutant ( uvrA, recA ) of Escherichia coli. In the E. coli mutant pyrimidine dimers are believed to be the primary cause of lethality at both wavelengths. Evidence for dimer involvement in the yeast mutant was obtained by demonstrating that lethality at both 254 and 365 nm was photoreactivated by light at 405 nm.     

MODIFYING FACTORS OF THE CELLULAR CONCENTRATION OF PHOTOLYASE MOLECULES IN Saccharomyces cerevisiae—II. EFFECTS OF PREILLUMINATION WITH LIGHT FLASHES

Abstract The effects of preillumination with photoreactivating light flashes before UV-irradiation on the number of photoreactivable complexes consisting of UV-induced DNA-damages and active photolyase molecules (N_PREact), on the fluence decrements, ΔD_PRE, that are obtained from two UV-survival curves without and with 1 flash photoreactivation and proportional to N_PREact were determined in haploid Saccharomyces cerevisiae cells. ΔD_PRE increased by preillumination from 0.115 Jm^–2 to 0.460 Jm^–2 and from 0.376 Jm^–2 to 0.494 Jm^–2 in cells in logarithmic growth phase and in stationary growth phase, respectively. Δ_PRF in log-cells that were preilluminated before and after resuspension in buffer at 40°C for 60 min was larger than ΔD_PRE in log-cells preilluminated only after resuspension in buffer.     

PHOTOMORPHOGENESIS IN Penicillium isariaeforme: EXOGENOUS CALCIUM SUBSTITUTES FOR LIGHT

Abstract— Penicillium isariaeforme is a photomorphogenic fungus which produces upright bundles of conidia-bearing mycelia (called coremia) when grown on a defined medium in visible (450–500 nm) light. We found that exogenous Ca²⁺ ions could substitute for light. In the dark 1–2 m M Ca²⁺ triggered coremia formation. Dark induction of coremia was specific for Ca²⁺ in that it could not be duplicated by 50 m M Ba²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Sr²⁺, or Zn²⁺. Additionally, light-induced coremia formation was inhibited by both KI (2.5 m M ) and phenylacetic acid (0.25 m M ).     

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.