COMPARISON OF SURVIVAL OF A U.V.-IRRADIATED URACIL-REQUIRING STRAIN OF ESCHERICHIA COLI B IN THE PRESENCE OF CHLORAMPHENICOL AND IN THE ABSENCE OF THE ESSENTIAL METABOLITE

Abstract— Restoration of viability of E. coli B U- after a total dose of 900 ergs/mm2 U.V. in the presence of chloramphenicol and in the absence of uracil was studied. Both treatments result in the recovery of the same fraction of the irradiated bacterial population. There is no additive effect if both treatments are applied to the same irradiated culture.     

DECREASED SURVIVAL RESULTING FROM LIQUID-HOLDING OF U.V.-IRRADIATED ESCHERICHIA COLI C AND SCHZZOSACCHAROMYCES POMBE

Abstract— Ultraviolet-irradiated cells of E. coli C and of haploid wild type yeast Schizosac-charomyces pombe , held in buffer at 22°-25°C for various periods of time prior to plating, show a lower survival than those plated immediately after irradiation. This 'negative liquid-holding effect' (NLHE) contrasts 'liquid-holding recovery' (LHR), found in a number of other E. coli strains and in Saccharomyces cerevisiae . NLHE was observed at all u.v. doses tested. The effect is maximal at holding temperatures in the range 25–30°C, it is very small at 5°C and (in E. coli C) at 44°C. NLHE and LHR resemble each other in several respects. In E. coli both effects are inhibitable by the dark repair inhibitors acriflavine, caffeine and potassium cyanide. They do not occur in nutrient broth, and they are much reduced if the irradiated cells were illuminated with photoreactivating light before holding. NLHE in S. pombe shows characteristics similar to those observed in E. coli C . Mutations leading to increased u.v. sensitivity in E. coli C and S. pombe can alter the liquid-holding response so that LHR is observed. Tetrad analysis of crosses between u.v.-sensitive and u.v.-resistant S. pombe strains indicates that a single chromosome region can control both u.v. sensitivity and liquid-holding response. Several possibilities explaining NLHE are discussed. From current knowledge about dark repair processes and from the similarities between NLHE and LHR in E. coli it seems likely that the two effects reflect slight changes in the efficiency of dark repair.     

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.     

U.V. INDUCED CONFORMATIONAL CHANGES IN TRANSFER RNA

Abstract— The quantum yields for the u.v. inactivation of the amino acid acceptor function of E. coli transfer RNA (for val, phe and lys) and for the loss of its conformation, as a function of exposure, have been determined following irradiation at 280, 265 and 254 nm. Our results suggest that u.v. damage produces a change in the conformation of transfer RNA which in turn inactivates it, and that the anticodon is not the u.v. sensitive site. Calculations indicate that a small number of photoproducts inactivate the transfer RNA.     

THE ROLE OF PEROXIDES IN THE INHIBITION OF DNA SYNTHESIS IN CELLS FOLLOWING IRRIDIATION WITH A U.V. MICROBEAM

Abstract Previous work had shown that the rate of DNA syntheses in mammalian cells could be reduced when a microbeam of ultraviolet light was directed into the medium surrounding the cells. The present work was designed to gather informtion on the mechanism of this effect.
Results show that catalase does protect the cellualar DNA synthesis under the following conditions.
(1) The microbeam must be directed into the surrounding medium—there is no protection if it strikes any part of the cell.
(2) The catalase must have been in contact with the cells for at least 4 hr before irradiation.
(3) At high closes of radiation the protection is only partial.
The catalase was labelled with fluorescent dye to demonstrate that it was able to penetrate the cell cytoplasm but not nucleus. Morphological studies on cell exposed to medium containing hydrogen pereoxide showed that protection was provided by intracellular and not extracellular catalasse and supported the idea that the toxic product produced by ultraviolet radiation of the medium was ultimately a peroxide.     

ULTRAVIOLET RADIATION-INDUCED MUTABILITY OF uvrD3 STRAINS OF ESCHERICHIA COLI B/r AND K-12: A PROBLEM IN ANALYZING MUTAGENESIS DATA

Abstract— The involvement of the uvrD gene product in UV-induced mutagenesis in Escherichia coli was studied by comparing wild-type and uvrA or uvrB strains with their uvrD derivatives in B/r and K-12(W3110) backgrounds. Mutations per survivor (reversions to prototrophy) were compared as a function of surviving fraction and of UV fluence. While recognizing that both methods are not without problems, arguments are presented for favoring the former rather than the latter method of presenting the data when survival is less than 100%. When UV-induced mutation frequencies were plotted as a function of surviving fraction, the uvrD derivatives were less mutable than the corresponding parent strains. The B/r strains exhibited higher mutation frequencies than did the K-12(W3110) strains. A uvrB mutation increased the mutation frequency of its parental K-12 strain, but a uvrA mutation only increased the mutation frequency of its parental B/r strain at UV survivals greater than ˜ 80%. Both the uvrA and uvrB mutations increased the mutation frequencies of the uvrD strains in the B/r and K-12 backgrounds, respectively. Rather different conclusions would be drawn if mutagenesis were considered as a function of UV fluence rather than of survival, a situation that calls for further work and discussion. Ideally mutation efficiencies should be compared as a function of the number of repair events per survivor, a number that is currently unobtainable.     

METABOLISM OF NUCLEIC ACIDS IN U.V. IRRADIATED L-STRAIN CELLS TREATED WITH NATIVE DEOXYRIBONUCLEIC ACID

Abstract— In our earlier papers we described the recovery of U.V. irradiated L-strain cells by means of nucleic acids. In an attempt to get better insight in the mechanism(s) leading to this recovery we studied the effect of native DNA on the metabolism of nucleic acids of recipient cells. In these experiments we have found that the label isologous DNA-C¹⁴ is incorporated in DNA of normal and irradiated recipient cells. The rate of DNA-C¹⁴ label incorporation in the DNA of irradiated recipient cells is slower than in the unirradiated controls. The presence of thymidine in the incubation media reduces the incorporation rate of DNA-C¹⁴. When the irradiated cells have been growing in a medium containing isologous DNA in presence of thymidine or heterologous DNA the recovery effect was not observed.     

ROLES OF THE RELA+ GENE AND OF 4-THIOURIDINE IN NEAR-ULTRAVIOLET (334 nm) RADIATION INHIBITION OF INDUCED SYNTHESIS OF TRYPTOPHANASE IN ESCHERICHIA COLI B/r

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) is known to inhibit the induced synthesis of tryptophanase by tryptophan in Escherichia coli , showing an action spectrum similar to that for near-UV-induced growth delay. The present work shows that a rel A mutant of E. coli B/r exhibits 50% as much monochromatic near-UV (334 nm) inhibition of tryptophanase induction as the wild type, and that a mutant lacking 4-thiouridine, an unusual nucleoside in tRNA, exhibits < 10% as much inhibition of tryptophanase induction. These findings indicate that 4-thiouridine is almost the sole chromophore for this effect in E. coli B/r, but that only 50% of the effect operates by a mechanism utilizing the rel A ⁺ gene product; growth delay appears not to be primarily involved.     

ACTION SPECTRUM FOR GROWTH DELAY INDUCED BY NEAR-ULTRAVIOLET LIGHT IN E. coli B/r K*

Abstract— The action spectrum for growth delay induced by near-uv light was determined for Escherichia coli B/r growing in a defined medium. This spectrum agrees with and extends that determined earlier by Jagger and his co-workers for E. coli B growing in nutrient broth. The extended spectrum is indistinguishable from the absorption spectrum for 4-thiouridine above 320 nm, but deviates significantly at wavelengths shorter than this from the spectrum for 8–13 link formation in transfer RNA containing 4-thiouridine at position 8. These results extend the evidence that 4-thiouridine in transfer RNA is the chromophore for near-UV induction of growth delay, but weaken the case for linkage of a pyrimidine at position 13 in transfer RNA as the mechanism of growth delay.     

PHOTOPROTECTION OF E. COLI B/r: RESPIRATION,GROWTH, MACROMOLECULAR SYNTHESIS AND REPAIR OF DNA*

Abstract— Photoprotecting effects of near UV radiations (300–400 nm, maximum at 360 nm) against far UV radiations (primarily 254 nm) have been studied in Escherichia coli B/r cells in minimal medium with glycerol as a carbon source. Near UV light (10⁵ Jm^-2) has a negligible effect on survival, but causes transitory inhibition of respiration, growth, DNA, RNA, and protein syntheses and cell division. Far UV (52 J m^-2) reduces survival to about 0.5 per cent; respiration, growth and RNA and protein syntheses proceed for about 60 min, after which they nearly cease for several hours. Near UV given before this fluence of far UV increases survival 10-fold and the above processes resume at times and with kinetics characteristic of those produced by lower fluences of far UV. Single-strand breaks appear in the DNA of both unprotected and photoprotected cells; repair of the breaks is essentially complete in protected but not unprotected cells. The viability kinetics for far-UV-irradiated cells with and without photoprotecting treatment are identical except that the curve for the latter is displaced upward about 1 log; exponential increases (cell division) begin at 120 min in each case. The data suggest that, in B/r cells grown under our particular conditions, namely in minimal medium with glycerol, photoprotection is not the result of growth or division delays, but reflects an increased repair capability due to continued respiration.     

SUPPRESSION OF PHAGE T4 BY CO-INFECTION WITH U.V.-INACTIVATED HOMOLOGOUS PHAGE

Abstract— Unirradiated phage T4v₁ may fail to produce viable progeny in cells which are co-infected with u.v.-irradiated homologous particles. The extent of this effect, called suppression , is positively correlated with the multiplicity of infection of the irradiated phage and with the U.V. dose. The suppression reaches a maximum level at about 30–600 lethal hits. Quantitative evaluation of the results shows that in some complexes one irradiated phage particle is sufficient to suppress an unirradiated phage. Two hypotheses are discussed to explain the results. (a) Lethal u.v.-damages are incorporated into the unirradiated phage genome by genetic recombination; ( b ) Genetic subunits (e.g. cistrons or operons) of the u.v.-irradiated phages produce informationally incorrect messenger RNA molecules, which compete with the correct ones from the unirradiated phage in the protein-synthetizing system. Hypothesis (6) appears to be more adequate to explain the experimental results.     

THERMOLUMINESCENCE OF NUCLEIC ACID CONSTITUENTS IN SOLUTION AFTER U.V.-IRRADIATION

Abstract— The thermoluminescence of purines and pyrimidines has been studied in ethylene glycol (EG)/water (2:1) glass matrices after ultraviolet (u.v.) irradiation at 77°K. An investigation of the mechanism of thennoluminescence has been made for adenine. The thermoluminescence involved biphotonic ionisation and recombination. The thermoluminescence depended on the square of the excitation light intensity, and the observed lifetime of the second photonabsorbing intermediate was the same as the phosphorescence lifetime. The emission spectrum lay in the same wavelength region as the phosphorescence spectrum. It was possible to bleach the thennoluminescence with visible light.     

IN VIVO INDUCTION OF 4-THIOURIDINE-CYTIDINE ADDUCTS IN tRNA OF E. COLI B/r BY NEAR-ULTRAVIOLET RADIATION*

Abstract— Near-ultraviolet (near-UV; 320–405 nm) irradiation of Escherichia coli B/r induces the formation in vivo of ⁴Srd-Cyd adducts in transfer RNA, as evidenced by (1) fluorescence spectrum changes of tRNA extracted from irradiated cells and reduced with NaBH₄, (2) thin-layer chromatography on cellulose of hydrolysates of trichloroacetic acid-precipitable extracts of irradiated cells, and (3) comparison of these findings with adduct formation induced by near-UV irradiation of purified mixed tRNA from E. coli. The kinetics of induction of the ⁴Srd-Cyd adduct in vivo, and the near-UV fluences required, provide strong support for our earlier hypothesis that formation of these adducts is responsible for near-UV-induced growth delay in E. coli.     

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.     

THE FUNCTION OF PHYTOCHROME IN THE NATURAL ENVIRONMENT–V. SEASONAL CHANGES IN RADIANT ENERGY QUALITY IN WOODLANDS

Abstract. The spectral distribution of radiant energy on the floor of three typical English woodlands (birch, beech and oak) has been monitored throughout the whole growing season. The results are presented in terms of the changes in the ratio of the photon fluxes at 660 nm and 730 nm (τ) and in the photosynthetically active radiation (PAR). The results are discussed in relation to the hypothesis that the role of phytochrome is to detect changed light quality due to mutual shading and to direct development accordingly.     

DELAY IN GROWTH AND DIVISION INDUCED BY NEAR ULTRAVIOLET RADIATION IN ESCHERICHIA COLI B AND ITS ROLE IN PHOTOPROTECTION AND LIQUID HOLDING RECOVERY

Abstract. Microscopic observations show that growth delay and division delay occur on nutrient agar after Escherichia coli B has been irradiated at 3341 Å. These effects also occur in nutrient broth.
A near u.v. action spectrum for growth delay in nutrient broth has been obtained. It shows a single peak at 3380 Å and is indistinguishable from the action spectrum for photo-protection from far u.v. (2537 Å) killing in the same organism. Furthermore, photoprotected cells show a much greater growth delay than cells that have not been photoprotected. These, as well as kinetic data, suggest that the essential action of a photoprotection treatment consists in the induction of a growth-division delay. This delay would presumably permit more time for intracellular recovery systems to operate on the far u. v. damage to nucleic acids.
Liquid holding recovery (effected by holding cells in phosphate buffer after far u. v. irradiation) shows complete overlap with photoprotection. It is concluded that photoprotection and liquid holding recovery operate on the same far u. v. damage. As with photoprotection, it is probable that the essential action of a liquid holding treatment is the induction of a growth-division delay.
No photoprotection is observed of intracellular T2 bacteriophage or of E. coli B_s-l (Hill).