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.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

A STUDY OF THE RELATIONSHIP BETWEEN SURVIVAL AND REPAIR SYNTHESIS'' OF DNA AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. and the postirradiation repair synthesis of E. coli 15 T^- 555-7 thy meth arg trp and E. coli B/r (HCR⁺) was followed. Prestarvation increased the number of survivors about 30–100 fold in both strains at doses 600-1200ergs/mm². In contrast to survival no increase in repair synthesis was observed. Thus, the increase in survival has to be brought about by a mechanism which seems to be independent of additional repair synthesis.     

EXCISION OF PYRIMIDINE DIMERS FROM THE NUCLEAR DNA OF A HAPLOID RESPIRATION-DEFICIENT (ρ) STRAIN OF SACCHAROMYCES CEREVISIAE

Abstract— A haploid respiration-deficient (ρ^-) mutant of Saccharomyces cerevisiae exhibits a lower ability to excise ultraviolet-induced pyrimidine dimers in various post-irradiation treatments as compared to the original respiration-competent wild-type strain. This decrease in excision may account for the inability of ρ^- strains to show liquid-holding recovery.     

SENSITIZED PHOTOREDUCTION OF CO(EDTA)- IN AERATED AQUEOUS SOLUTION. ITS PHOTO-ELECTROCHEMICAL AND PHOTOBIOLOGICAL APPLICATIONS

Abstract— The reduction of Co(edta)^- in aerated aqueous solution containing 1-hydroxy-5-oxo-5H-pyrido[3,2a]phenoxazine-3-calboxylic acid (HPPC) as sensitizer and ethylenediaminetetraacetate ion(EDTA) as electron donor was studied. Semireduced HPPC, formed in a reaction between triplet HPPC and EDTA, was shown to reduce Co(edta)^- to Co(edta)^2-. The ratio of the rate constant for oxidation of semireduced HPPC by oxygen to that by Co(edta)^- was determined to be 0.45± 0.01 from competition reaction kinetics. A (concentration)half-cell (oxidation-reduction electrode) based on the photoreduction of Co(edta)^- was found to give an electrode potential decrease of 100 mV. Co(edta)^- was also found to be reduced during illumination in aerated suspension of chloroplasts. The rate of oxygen evolution for the Co(edta)^- -chloroplast system was nearly the same as that for the Fe(CN)₆^3- -chloroplast system both in the absence and presence of ammonium ions.     

THE ROLE OF HOST-CELL REPAIR IN LIQUID-HOLDING RECOVERY OF U.V.-IRRADIATED ESCHERICHIA COLI

Abstract— The experiments reported give evidence that liquid-holding recovery (LHR) of u.v. irradiated E. coli cells involves basically the same type of dark repair which causes reactivation of phage and which results in much increased survival of the cells themselves [host-cell reactivation (HCR)]. LHR is very small in the two HCR(-) strains B syn^- and B_s-1, but occurs to larger but different extents in the three HCR(+) strains B, B/r, and B/r (Λ). LHR is inhibited if the liquid contains caffeine or acriflavine, both of which are known to inhibit HCR. The results indicate that most of the LHR effect, if not all, occurs during the liquid holding, rather than under growth conditions after liquid holding. It is assumed that the holding itself allows a prolonged time for, and therefore an enhancement of, HCR. It is thus implicit that LHR can be observed only where otherwise HCR of repairable u.v. damage would be incomplete, and that different extents of LHR, as observed in the three HCR(+) strains, reflect different extents of incompleteness of HCR. It is concluded that the repairable u.v. hits which are not fully repaired by HCR are predominantly those concerned with the extra u.v. sensitivity of the strains B and B/r (Λ), relative to B/r.     

EFFECT OF PHOTOCROSSLINKING ON ESCHERICHIA COLI tRNA STRUCTURE

Abstract— The effect of photocrosslinking s ⁴ U ₈ with C ₁₃ on the structure of E. coli tRNA has been examined by high resolution NMR. Photocrosslinking affects both the structure and the enzymatic acylation of tRNA. The NMR measurements demonstrate that except for the s ⁴ U ₈.A₁₄ base pair, there is no evidence that photocrosslinking perturbs any of the common tertiary structure interactions. However, this modification does perturb several resonances which are assigned to the secondary structure base pairs of the hU stem and the terminal base pair of the acceptor stem. The variable effect of photocrosslinking on the extent and rate of enzymatic acylation of different tRNA is attributed to different recognition sites for tRNA by the cognate synthetases.     

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.     

CHLORIDE ENHANCEMENT OF QUENCHING OF TRIPLET METHYLENE BLUE BY Fe(II)

Abstract— The influence of chloride ion on the rate of decay of triplet methylene blue in 0.01 M acid in the absence and presence of ferrous ions was investigated by means of laser flash-photolysis monitored by kinetic spectrophotometry. Chloride weakly accelerates decay of ³MBH^2± in aqueous solution in the absence of Fe(II). Quenching of ³MBH²⁺ by Fe(II) is more strongly catalyzed by Cl^- in both water and 50 v/v% aq. CH₃CN. The uncatalyzed quenching constant, k ₅, is of the order of 1 × 10⁶ M ^-1 s^-1 while in 4.8 M aqueous chloride ( μ – 7.2 M ) k ₅= (37.2 ± 1.8) × 10⁶ M ^-1 s^-1. A possible role of chloride is as a bridging species in quenching via electron transfer between ³MBH²⁺ and Fe(II).     

MISCHKLONE U.V.-INDUZIERTER MUTANTEN BEIM PHAGEN KAPPA

Abstract— Clear-plaque mutations of phage k of Serratia are induced by extracellular u.v.-irradiation in a 2–hit process. The 2–hit-nature cannot be due to induction of one hit in each of the two DNA-strands since replating the contents of 97 wildtype plaques of u.v.-survivors (u.v.-dose 4.5 min) revealed only 1 case of heterozygosity; at least 20 cases would have been expected if phage with 1–hit-mutations formed phages looking like wildtype. On the other hand, only 1 case of heterozygosity was observed among replatings of 94 c -mutants induced by the u.v.-dose 4.5 min (survival 10^-3); most of the plaques contained pure c -type. The pure c -mutant clones are very probably due to 'recessive' lethal lesions in the nonpremutated DNA-strand. This is indicated by the dose dependence of the frequency of heterozygotes; at a dose of 3 min u.v. (survival 1.2 × 10^--²) 9 heterozygotes were observed among 95 mutants tested. From these numbers the rate of induction by u.v. of the recessive lesions's can be calculated. The data at the higher dose are in satisfactory agreement with the calculated rate. Also other types of plaque mutations (e, t, b ) showed heterozygosity. Two cases of abnormal heterozygosity were observed; one contained 2 stable mutant types ( c and b ), another one wildtype, c -tm and l -type.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

THE U.V. SENSITIVITY OF BACTERIA: ITS RELATION TO THE DNA REPLICATION CYCLE

Abstract— A striking increase in the shoulder of the u.v. survival curve but no change in the limiting slope is obtained when cultures of Escherichia coli strain TAU complete the DNA replication cycle in the absence of concommitant protein synthesis prior to irradiation. The u.v. sensitivity of protein synthesis or RNA synthesis is not altered significantly by this treatment.
In contrast to the result for strain TAU, there is no significant change in the u.v. survival curve for the u.v. sensitive E. coli B_s-1 when its DNA replication cycle is completed under similar conditions.
Following a period of inhibited protein synthesis there is a delay in the reinitiation of the normal DNA replication cycle when protein synthesis resumes. This delay would allow time for an intracellular repair system to operate before the attempted resumption of normal replication. Strain B_s-1, which is deficient in this repair system, would not be expected to benefit from such a delay, as consistent with the observed results. A model is presented to account for lethality due to attempted DNA replication during a period of repair synthesis. The maximum survival for a given u.v. dose would be predicted for a culture which has completed the normal DNA replication cycle prior to irradiation and which is not permitted to reinitiate the cycle until all possible repair synthesis is completed.     

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.     

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.     

CHANGES IN U.V. SURVIVAL CURVES OF ESCHERICHIA COLl B/r CONCOMITANT WITH CHANGES IN GROWTH CONDITIONS

Abstract— For E. coli B/r u.v.-irradiated while in logarithmic growth, the nature of the dose-response curve was strongly dependent on both pre- and post-irradiation conditions of growth. Survival curves for cells grown in nutrient medium, or minimal medium with glucose, and plated immediately after irradiation, demonstrated an initial insensitive or 'shoulder' region provided the plating medium was such that no derepression was required of operons controlling inducible enzyme systems. If, however, such derepression was called for, survival curves were of exponential form. Delay in plating resulted in the return of the survival curve to the shouldered form even when 'shift-down' media were used.
Of those cells grown before u.v.-irradiation in minimal media and plated thereafter with the same sugar as carbon source, only those grown with glucose (or lactose) demonstrated the shouldered survival curve.     

HYDROGEN PEROXIDE INDUCED RESISTANCE TO BROAD-SPECTRUM NEAR-ULTRAVIOLET LIGHT(300–400 nm) INACTIVATION IN Escherichia coli

Abstract— Strains of Escherichia coli carrying the four possible combinations of the alleles nur, nur⁺, uvrAb, and uvrA ⁺ were either untreated or pretreated with a sublethal dose of H₂0₂ prior to inactivation with NUV radiation. Pretreated cells exhibited a greater resistance to NUV than did untreated cells. Pretreatment with H₂O₂ did not induce resistance to FUV radiation. The induction of resistance to NUV inactivation by H₂O₂ pretreatment apparently leads to protection against the damage caused by NUV radiation. Although pretreatment of cells with H₂0₂ leads to resistance of such cells to inactivation by H₂O₂ and NUV, survival of H₂O₂ treated bacteriophage PI cml clr100 is not enhanced when assayed on H₂O₂ pretreated E. coli host cells.     

CARBOXYLATION OF FORMIC ACID IN AQUEOUS SOLUTIONS UNDER THE INFLUENCE OF U.V.-LIGHT

Abstract— Carboxylation of various organic substances by dissolved carbon dioxide or bicarbonate is achieved by the action of U.V. -Light of 184.9 and 253.1 mμ on water. The transformation of formic acid to oxalic acid was investigated in more detail as a function of u.v.-dose, pH-values and the formate concentration. For an irradiation period of 12 hr of 0.1 M formate, a yield of about 2 m mol.1^--¹ oxalic acid was obtained. A reaction mechanism for the photochemical carboxylation process is presented. The results are of some interest in respect of the synthesis of organic compounds under the primitive earth conditions.     

SOLID PHASE PHOTOREDUCTION OF METHYLVIOLOGEN ON CELLULOSE SENSITIZED BY TRIS(2,2'-BIPYRIDYL)RUTHENIUM COMPLEX

–Methylviologen (MV²⁺) adsorbed on cellulose could be reduced photochemically in the solid phase sensitized by tris(2,2'-bipyridyl)ruthenium(II) complex, [Ru(II)(bpy)₃], using disodium ethylene-diaminetetraacetic acid (Na₂EDTA) as a reducing agent. Formation of the cation radical (MV ⁺.) was confirmed by visible and EPR spectra. The MV⁺. formed on cellulose was remarkably stable against air oxidation and rapidly accumulated even by the irradiation under air. Water adsorbed on the cellulose greatly retarded the photoreaction. Action spectrum showed that the excitation of Ru(II)(byp)₃ is responsible for the photochemical reaction. The quenching of the emission from Ru(II)(bpy)*₃ by MV²⁺. indicated that a primary photochemical reaction occurs between Ru(II)(bpy)*₃ and MV²⁺. The main reaction path is the reduction of MV²⁺ by Ru(II)(bpy)₃, giving MV⁺. and Ru(III)(bpy)₃, followed by the reduction of Ru(III)(bpy)₃ to Ru(II)(bpy)₃ with Na₂EDTA, which in turn is oxidized to give carbon dioxide.     

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.     

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.).