CHANGE IN THE BASE COMPOSITION OF MESSENGER RNA OF ESCHERICHIA COLI BY ULTRAVIOLET IRRADIATION AND ITS RECOVERY

Abstract— The base composition of messenger RNA in Escherichia coli B/r and B _8–1 irradiated with ultraviolet (u.v.) light has been examined. The experimental results are as follows: (1) the synthesis of rapidly labeled RNA does not stop in ultraviolet irradiated bacteria. (2) The rapidly labeled RNA in irradiated cells shows a change in base composition corresponding to the formation of pyrimidine dimers in DNA molecules. The mole per cent of adenine component is increased with ultraviolet dose. The ratio of purine/pyrimidine becomes larger and the GC content smaller. (3) The base composition of the rapidly labeled RNA in irradiated bacteria reversed to that in unirradiated cells, when the irradiated cells were reactivated by experimental procedures for photoreactivation or dark reactivation. The reversion in the base composition corresponds well to the decrease in the amount of thymine dimers in DNA molecules. (4) The mechanism of the change in the base composition of rapidly labeled RNA caused by ultraviolet irradiation is discussed.     

LETHAL EFFECTS OF NATURAL SOLAR ULTRAVIOLET RADIATION IN REPAIR PROFICIENT AND REPAIR DEFICIENT STRAINS OF ESCHERICHIA COLI: ACTIONS AND INTERACTIONS

Abstract— The inactivation of repair proficient ( Escherichia coli K12 AB 1157, E. coli B/r) and repair deficient ( E. coli K12 AB 1886 uvrA , AB 2463 recA and AB 2480 uvrA recA ) strains of bacteria by noon sunlight has been measured. The use of biological dosimetry based on an ultraviolet (UV) sensitive strain of Bacillus subtilis spores has allowed a quantitative comparison of bacterial inactivation by solar, 254 and 302 nm radiations. Our analysis indicates that: (1) uvrA and recA gene products are involved in repair of a substantial portion of the solar DNA damage, (2) 302 nm is a more appropriate wavelength than 254 nm to represent the DNA-damaging action of sunlight and that (3) repair proficient strains are inactivated by sunlight more rapidly than expected from the levels of DNA damage induced. When populations of repair proficient bacteria are exposed to noon sunlight for 20 min, they become sensitive to the lethal action of far-UV (254 nm), MMS (0.1 M ) and to a lesser extent, mild heat (52°C).     

THE SYNERGISTIC ACTION OF ULTRAVIOLET AND X RADIATION ON MUTANTS OF ESCHERICHIA COLI K-12

Abstract— Prior UV irradiation increased the X-ray sensitivity of wild-type E. coli K-12. This synergistic effect of combined UV and X irradiation was also observed, but to a reduced extent, in uvrA, uvrB, uvrC , and polA mutants, but was absent in exrA, recA, recB , or recC mutants of E. coli K-12. Alkaline sucrose gradient studies demonstrated that the wand err gene-controlled, growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks was inhibited by prior UV irradiation. This inhibition probably explains the synergistic effect of these two radiations on survival.     

INACTIVATION OF THE LACTOSE PERMEASE OF ESCHERICHIA COLI BY MONOCHROMATIC ULTRAVIOLET LIGHT

Abstract— The lactose permease of E. coli is inactivated exponentially by seven wavelengths of monochromatic UV light. An action spectrum reveals that the shorter wavelengths (243, 290 and 313 nm) are much more efficient than longer wavelengths. Inactivation at 290 nm is most efficient and is not due to generalized membrane damage. The rate of counterflux of intracellular β-galactoside in response to externally added β-galactoside was slowed by 290 nm irradiation, indicating destruction of the facilitated diffusion mechanism. The induction of β-galactosidase and β-galactoside permease was co-ordinate both with and without pre-irradiation by 290 nm light. The β-galactosidase is approximately 26-fold more resistant to 290 nm than the permease. These results are discussed in terms of a greater sensitivity of membrane proteins to 290 nm light, which may be due to the role of aromatic amino acids in conferring stability to the permease in the membrane.     

SOME OBSERVATIONS ON THE LETHAL EFFECTS OF NEAR-ULTRAVIOLET LIGHT ON ESCHERICHIA COLI, COMPARED WITH THE LETHAL EFFECTS OF FAR-ULTRAVIOLET LIGHT

Abstract— Near-ultraviolet light (365.5 nm) reduces the ability of Escherichia coli B/r and B_8-1, to form colonies on nutrient agar after irradiation. This lethal effect is distinct from that obtained after far-u.v. irradiation (253.7 nm) because the far-u.v. sensitive and resistant strains are equally susceptible to near-u.v. Variation in susceptibility to ultraviolet light during growth is more marked for near-u.v. than for far-u.v. The number of survivors after near-u.v. irradiation of log phase cells is affected by several post-irradiation treatments; more cells survive if growth immediately after irradiation occurs at higher temperatures (unlike far-u.v.). Also, the presence of acriflavine and caffeine in the nutrient agar decreases the number of survivors (in common with far-u.v.).     

PHOTODYNAMIC INHIBITION OF ESCHERICHIA COLI DNA POLYMERASE I BY 8-METHOXYPSORALEN PLUS NEAR ULTRAVIOLET IRRADIATION

Abstract— Morphological abnormality due to the UV irradiation of sperm and its modification by photoreactivation (PR) were studied in the sea urchin, Hemicentrotus pukherrimus. When sperm was UV-irradiated and allowed to fertilize unirradiated eggs, the effect of the UV was manifested as an abnormal morphology of embryos in the gastrula or later stages. The UV-induced morphological abnormality was prevented by photoreactivation when the fertilized eggs were illuminated with visible light. In the experiments on a stage-dependent change of PR effectiveness, it was found that an illumination sufficed to effect a nearly complete PR when applied up to the onset of the first DNA synthetic phase, while the PR effectiveness declined thereafter. Illumination after the completion of DNA synthesis had little effect for PR.     

SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

ENHANCED SENSITIVITY TO THE LETHAL AND MUTAGENIC EFFECTS OF PHOTOSENSITIZING ACTION OF CHLORPROMAZINE IN ETHYLENEDIAMINETETRAACETATE-TREATED ESCHERICHIA COLI

Abstract— Ethylenediaminetetraacetate (EDTA) treatment of Escherichia coli H/r30 (Arg_-) enhanced cell sensitivity to the lethal and mutagenic effects of the photosensitizing action of chlorpromazine (CPZ). The most obvious effect of EDTA on the fluence-survival curve was an elimination of the shoulder. In the absence of EDTA, CPZ plus near-UV radiation did not induce the reversion from arginine-auxo-troph to autotroph of E. coli H/r30. However, when EDTA (5 mM)-treated cells were subjected to CPZ plus near-UV radiation, the induced reversion frequency increased with time of irradiation. It is concluded that the enhanced penetration of CPZ into E. coli cells by EDTA facilitates the drug binding to DNA within the cells upon near-UV irradiation and that this is the cause for the enhanced photosensitized lethal and mutagenic effects of CPZ.     

CONDITIONS AFFECTING THE EARLY THYMINELESS DEATH OCCURRING AFTER ULTRAVIOLET IRRADIATION OF ESCHERICHIA COLI B3

Abstract— Exposure of the thymine requiring bacterium Escherichia coli strain B3 to ultraviolet light (u.v.) prior to incubation in the absence of thymine shortens the lag period normally observed before the onset of death due to lack of thymine. Culture conditions promoting synthesis of new kinds of enzymes at the time of thymineless challenge after u.v. irradiation enhance this effect. The effect can be reversed either by the addition of thymine or photo-reactivation. Possible mechanisms for these phenomena are discussed.     

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON ESCHERICHIA COLI MEMBRANES AND DNA IN VIVO

Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in rec A, uvr A, or pol A functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was provided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-ß-D-galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.     

PROTECTION AGAINST ULTRAVIOLET B RADIATION-INDUCED EFFECTS IN THE SKIN OF SKH-1 HAIRLESS MICE BY A POLYPHENOLIC FRACTION ISOLATED FROM GREEN TEA

In prior studies we and others have shown that oral feeding of a polyphenolic fraction isolated from green tea (GTP) or water extract of green tea affords protection against ultraviolet B (UVB) radiation-induced carcinogenesis in SKH-1 hairless mice (Wang et al., Carcinogenesis 12, 1527–1530, 1991). It is known that exposure of murine skin to UVB radiation results in cutaneous edema, depletion of the antioxidant-defense system and induction of ornithine decarboxylase (ODC) and cyclooxygenase activities. In this study we assessed the protective effect of GTP on these UVB radiation-caused changes in murine skin. Oral feeding of 0.2% GTP (wt/vol) as the sole source of drinking water for 30 days to SKH-1 hairless mice followed by irradiation with UVB (900 mJ/cm²) resulted in significant protection against UVB radiation-caused cutaneous edema ( P <0.0005) and depletion of the antioxidant-defense system in epidermis ( P <0.01–0.02). The oral feeding of GTP also resulted in significant protection against UVB radiation-caused induction of epidermal ODC ( P <0.005–0.01) and cyclooxygenase activities ( P <0.0001) in a time-dependent manner. Our data indicate that the inhibition of UVB radiation-caused changes in these markers of tumor promotion in murine skin by GTP may be one of the possible mechanisms of chemopreventive effects associated with green tea against UVB-induced tumorigenesis. The results of this study suggest that green tea, specifically polyphenols present therein, may be useful against inflammatory responses associated with the exposure of skin to solar radiation.     

PROTECTION BY THE FLUORIDE ION AGAINST PHTHALOCYANINE-INDUCED PHOTODYNAMIC KILLING OF CHINESE HAMSTER CELLS

When a dilute F- solution was added to a culture of Chinese hamster cells that had been preincubated with an aluminium phthalocyanine sensitizer derived from AlPcCl, the photosensitivity of the cells was markedly reduced compared to control cells not treated with F-. Under the same treatment conditions, the reduction in [3H]thymidine incorporation into cellular DNA caused by light and this sensitizer and the production of DNA-protein crosslinks caused by light and this sensitizer were also inhibited by F-. In contrast, the killing of Chinese hamster cells, the reduction of thymidine incorporation by the cells, and the production of DNA-protein crosslinks in the cells caused by the combination of light and either Photofrin II or the silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3-N(CH3)2 were not inhibited by F-. We conclude that the aluminium phthalocyanine sensitizer used is largely or completely AlPc(OH)(H2O), that it is converted to a fluoro complex by F-, and that this compound probably is a less efficient generator of photochemical damage at a critical cellular target(s) than is AlPc(OH)(H2O). The inhibition of thymidine incorporation and DNA-protein crosslink formation indicates that the effects of F- can be expressed at intracellular sites. It is further concluded that the silicon phthalocyanine sensitizer and Photofrin II do not interact significantly with F-.     

THE EFFECTS OF ULTRAVIOLET AND VISIBLE RADIATION ON MOUSE ASCITES TUMOUR CELLS

Abstract— Effects of ultraviolet and visible radiation on the viability of Landschutz ascites tumour cells have been tested by growing control and treated tumour samples in adult mice. The tumour cells were irradiated as a dilute suspension in isotonic buffered salt solution, and were equilibrated at 0°C with oxygen or with nitrogen before irradiation.
Tumour cell proliferation was measured by a variety of techniques. The preferred assay-method was the growth of solid tumours in the axillae and groins of mice after sub-cutaneous inoculation of varying dilutions of treated or control ascites tumour cells. The immune response of the mice to the injected cells was reduced by whole body irradiation with a 300r dose of x-rays two days before inoculation. Results were calculated from parallel line assays using the reciprocal of the delay in appearance of the solid tumours up to 30 days post-innoculation. This reciprocal (1/T) was linearly related to the logarithm of the number of cells inoculated.
Photoreactivation has been demonstrated for this system, in which both U.V. and visible radiations were absorbed by the same cells. Light delivered alone in oxygen or in nitrogen was without effect on cell-viability, but it increased cell-survival after u.v.-irradiation in nitrogen and decreased survival after u.v.-irradiation in oxygen. Ultraviolet radiation alone was not significantly more lethal in oxygen than in nitrogen. A further observation in this work was an interaction between irradiated and control tumour cells injected into the same animal.
It is suggested that the radiation used may affect the antigenic character of the tumour cells as well as their reproductive capadity.     

RESPIRATION RESPONSES OF A polA1 AND A tif-1 MUTANT OF ESCHERICHIA COLI TO FAR-ULTRAVIOLET IRRADIATION*

Abstract— Cessation of respiration in Escherichia coli 60min after far-ultraviolet (254 nm) irradiation is dependent upon the recA and lexA gene products and is regulated by cyclic 3′,5′-adenosine monophosphate (cAMP) and its receptor protein. Respiration responses to UV irradiation were studied in two E. coli B/r mutants, polA1 and tif-1, both of which express other rec/lex functions (such as mutagenesis) after UV irradiation. The cells were grown on glycerol minimal medium supplemented with required amino acids. After receiving a relatively high UV fluence, the polA1 mutant, deficient in DNA polymer-ase I, showed a respiration shutoff response like the wild type cells. 5-Fluorouracil and rifampin, an inhibitor of initiation of RNA synthesis, did not prevent respiration shutoff in the mutant cells as they did in the wild type cells. Thus, RNA synthesis is not necessary for cessation of respiration in polA1 cells and the process is not an induced one. At lower fluences which did not shut off respiration of polA1 cells, cAMP did not cause a more complete shutoff as it did for the wild type cells. The tif-1 mutant has a modified recA protein, and when unirradiated cells are incubated at 42°C they form filaments, mutate, and show other rec/lex responses. This mutant did not shut off its respiration at either 30 or 42°C, and the response was not modified by cAMP. An E. coli K12 strain, W3110, was also tested for its respiration response to UV. At 52J/m² respiration did not shut off and cAMP had no effect.     

THE ROLE OF PYRIMIDINE DIMERS AND NON-DIMER DAMAGE IN THE INACTIVATION OF ESCHERICHIA COLI BY UV RADIATION

Abstract— We have quantitated the role of pyrimidine dimers and non-dimer damage in the inactivation of Escherichia coli by far-UV radiation, near-UV radiation, and triplet state sensitized near-UV radiation. The extent of photoreactivation in vivo of an excision and postreplication repair-deficient strain of E. coli after the different radiation treatments has been correlated with the relative proportion of pyrimidine dimers and non-dimer lesions produced. Using an excision deficient strain of E. coli, the susceptibility to recA ⁺ -dependent repair of the damage produced by the different radiation treatments has also been quantified.     

THE ROLE OF 4-THIOURIDINE IN LETHAL EFFECTS AND IN DNA BACKBONE BREAKAGE CAUSED BY 334 nm ULTRAVIOLET LIGHT IN Escherichia coli

Strains of Escherichia coli that lack 4-thiouridine (⁴Srd) are killed by monochromatic 334 nm UV light (UV) less efficiently than their wild-type parents, which contain ⁴Srd. Oxygen enhancement ratios (OER) at 10% survival are 3.3 for a strain that possesses ⁴Srd, and 2.6 for one that lacks ⁴Srd. Single-strand breaks in DNA caused by 334 nm UV accumulate more than twice as fast in the wild-type strains than in the strains lacking ⁴Srd. The results suggest that ⁴Srd is an important chromophore in some near-UV lethal effects. The results also suggest that the excitation energy from 334 nm UV light may be passed from RNA to DNA, resulting in single-strand breaks.     

OXYGEN EFFECT IN THE PROTECTION OF E. COLI AGAINST U.V. INACTIVATION AND MUTAGENESIS BY ACRIDINE ORANGE*

Abstract— Protection by acridine orange against ultraviolet light effects in resting cells of E. coli B/r/1, try^- was studied with special reference to a possible oxygen effect. Dose-response relationships were described by the function S= 1–(1 - e-^kD)ⁿ where S is the surviving fraction and D is the u.v. dose in ergs/mm². For cells suspended in 5 × 10^--⁶M acridine orange (AO) in air, the radiation sensitivity k was reduced from 0.010 (ergs/mm²)^)-1 in the absence of the dye to 0.0053 (ergs/mm²)^-1 in the presence of the dye. Under anoxia at this AO concentration, k was further reduced to 0.0015 (ergs/mm²)^-1. The oxygen effect ratio, kO₂/k_N2, was 3.5 at this concentration of AO. Greater protection was observed in cells suspended in 2 × 10^--⁵M AO, the oxygen effect ratio was unchanged. No oxygen effect was detected in the u.v. response in the absence of the dye. The value of n was reduced from about 12 with no dye to about 5 at dye concentrations of 5 × 10^--⁶M AO or more when oxygen was present. Under anoxia, in the presence of AO, n was further reduced to about 1.3. Atebrin, an efficient u.v. protective agent but an inefficient photodynamic agent, had no oxygen effect for protection against u.v. inactivation. Acridine orange protected against u.v.-induced reversion to tryptophan indepence in E. coli WP2 to about the same extent as it did for inactivation. A similar oxygen effect was observed for both inactivation and mutagenesis.     

DEGRADATION OF THE DNA OF ESCHERICHIA COLI K12 REC- (JC1569b) AFTER IRRADIATION WITH ULTRAVIOLET LIGHT*

Abstract— Degradation of the DNA of a rec^- mutant of Escherichia coli K12 (JC1569 b) induced by u.v. light was investigated. The rate of degradation was much larger by growing bacteria than by stationary cells. When growing bacteria were starved for amino acids, their DNA became resistant to irradiation. The mode of u.v.-induced degradation was investigated by comparing the time course of release from the acid-insoluble fraction of the label for two growing cultures; the one was pulse-labeled with ³H-thymidine and the other was pulse-labeled and chased thereafter for 12 min. It was found that the label incorporated into the former culture begins to be lost from the acid-insoluble fraction prior to the loss of the label incorporated into the latter culture. It was concluded that breakdown of the replicating point precedes degradation of the bulk of the DNA. This result suggested that the replicating point is a sensitive site to irradiation and the u.v.-induced degradation of DNA seemed to be influenced by the state of chromosome at the time of irradiation. Experiments of centrifugation of lysed spheroplasts of bacteria uniformly labeled with ³H-thymidine in alkaline sucrose demonstrated that DNA of low molecular weight appeared after irradiation with only 5 ergs/ mm², and that the molecular weight could not be restored by post-irradiation incubation. Considering these results, an hypothesis is proposed concerning the initiation of induced degradation of the DNA of the rec^- mutant.