LIGHT-INDUCED ACCUMULATIONS OF DICTYOSTELIUM DISCOIDEUM AMOEBAE

Abstract— Light-induced accumulations of amoebae of Dictyostelium discoideum in 'light traps' have been observed. The greatest accumulations are obtained with cell densities of about 8 × 10⁴ cells/mm². Accumulations are observed at incident fluence rates over about one decade both for white and for monochromatic light; higher fluence rates cause dispersal from the 'light trap'. An action spectrum for the photoaccumulation, calculated from fluence-response curves using the zero thresholds, shows a major peak between 405 and 410 nm and extends through most of the visible spectrum. This action spectrum does not coincide with any of the pigments known to be present in D. discoideum . The cellular basis for the photoaccumulation has been studied. No light effects on cell divisions or cell aggregation are observed during the 2 h duration of an accumulation experiment. Microvideographic analysis of single amoebae is consistent with the hypothesis that the amoebae are positively phototactic and move toward the light scattered from cells in the 'light trap', thus accumulating in the trap.     

EFFECTS OF CAFFEINE ON DNA REPAIR OF UV-IRRADIATED DICTYOSTELIUM DISCOIDEUM

Abstract— Caffeine enhances the UV-killing of amoeboid cells of NC-4, but UV-irradiated γs-13 is insensitive to caffeine. UV-irradiated NC-4 becomes insensitive to the effect of caffeine during a postirradiation incubation in buffer for about 90 min, but γs-13 remains unchanged in the sensitivity to caffeine throughout the incubation for 180 min. Amoeboid cells of γs-13 can remove pyrimidine dimers as well as NC-4 even in the presence of caffeine. Caffeine inhibits rejoining of strand-breaks of DNA in UV-irradiated NC-4, but the rejoining in γs-13 is insensitive to caffeine.     

INHIBITION OF UV-INDUCED DNA STRAND BREAKAGE IN DICTYOSTELIUM DISCOIDEUM BY 2,4–DINITROPHENOL

Abstract— In UV-irradiated vegetative cultures of the cellular slime mold Dictyostelium discoideum NC-4, single strand breaks appeared in the DNA very rapidly and at low temperatures (0–4°C). However, when these cells were incubated, prior to UV irradiation, in the presence of 2 m M 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, breaks did not appear in the DNA. Extracts prepared from cells that had been incubated either in the presence or absence of DNP were tested for endonucleolytic activities on a UV-irradiated exogenous DNA template (φX-174 RF I). Results suggested that DNP might mediate its effect by interfering with the action of a UV-specific endonuclease.     

PHYSIOLOGICAL EFFECTS OF ULTRAVIOLET LIGHTON DICTYOSTELIUM DISCOIDEUM SPORE GERMINATION

Abstract— The spore germination in Dictyostelium discoideum consists of four stages: activation, postactivation lag, swelling and emergence. Ultraviolet irradiation (total fluence of 250 J/m²) of spores at any time prior to late spore swelling allows full swelling, but inhibits the emergence of myxamoebae. In the case of freshly activated spores, a UV exposure time of 30 s (total fluence of 50 J/m²) is sufficient to reduce emergence to about 6% when measured after 24 h of incubation. This same fluence results in about 10% viability as measured by plaque forming ability. Experiments utilizing 'fractionated exposures' result in the same percentage inhibition of emergence as that found for 'single exposures' provided the total fluence is equivalent. The higher fluences (250 J/m²) which completely prevent emergence, do not affect the endogenous oxygen uptake of spores during swelling. Ultraviolet light irradiated spores respond to the same activation and deactivation treatments as control unirradiated spores. Ultraviolet irradiation after late spore swelling allows emergence to occur in only a small fraction of the population. This fraction of cells which can emerge after UV treatment is said to have passed a 'competence point', which is believed to be the time when all the events necessary for emergence have been completed. Though the sites of UV inactivation in spores can only be postulated at present, it is apparent that the initial stages of germination (activation, postactivation lag and spore swelling) occur independently of the UV sensitive sites. The final stage of germination (emergence), however, is dependent on UV sensitive functions.     

INHIBITION OF DNA REPAIR SYNTHESIS BY SUNLIGHT

Abstract— DNA repair synthesis as determined by thymidine incorporation in the presence of hydroxyurea reached a much lower maximum level after solar compared with UVC exposure in five human melanoma cell lines, in HeLa cells, and in two human fibroblast strains. This finding was confirmed by determination of unscheduled DNA synthesis where both the number of labelled nuclei and grain count per nucleus were lower in sun-exposed cells. In a cloned human melanoma line (MM253cl), glass-filtered sunlight inhibited UVC repair synthesis, and solar UVB alone induced a higher level of repair synthesis than either complete sun or solar UVA plus solar UVB. The fluence response of filtered sunlight for inhibition of UVB (sunlamp) and UVC showed that most inhibition was obtained at low fluences (5-10 min), further exposure giving a plateau at 40% of the original level. Ultraviolet C and sunlight inactivated adenovirus 5 giving F ₀ values for virus survival 40-fold higher than for cell survival. Replication of either UVC- or solar-irradiated virus was not affected by prior irradiation of cells with glass-filtered sunlight. Stathmokinetic analysis of cell cycle progression by DNA flow cytometry showed that UVC and sunlamp UVB retarded cell movement from the G1 and S phases whereas equitoxic sunlight and glass-filtered sunlight (nontoxic) had no effect. These results indicate that solar UVA at low environmental fluences partially inhibits UVB repair synthesis in a range of human cell types but does not affect the replication of a UVB- or UVC-damaged virus when applied to the genome alone or to the host cell.     

LIGHT-INDUCED CAROTENE SYNTHESIS IN MUTANTS OF PHYCOMYCES WITH ABNORMAL PHOTOTROPISM

Mutants of Phycomyces. abnormal in their phototropic responses (the mad mutants), have been tested for their responses in light-induced carotene synthesis (LICS). The amount of carotene synthesized at any given fluence is significantly lower in the madA, madB and madD mutants than in the wild type. The amount of carotene is not lower in other mad mutants ( madC, madE, madF and madG ). The double mutant mad A madB and the triple mutant mad A madB madC show stronger effects. The wild type strain, as well as those carrying a single mad mutation ( madA and madB ) or those carrying two or three mad mutations ( madA madB. madA madB madC ) show closely similar sensitivity to LICS. This contrasts with phototropism and photoinitiation of sporangiophores which are sensitive to extremely small signals in the wild type and in which the madA mutation decreases the sensitivity by nearly a factor of 10⁴ and madB mutation by a factor of 10⁵. It appears that LICS does not share the signal amplification mechanisms characteristic of the other two responses.     

ULTRAVIOLET EFFECTS ON KILLING, FRUITING BODY FORMATION AND THE SPORES OF DICTYOSTELIUM DISCOIDEUM

Abstract— -Ultraviolet effects on amoeboid cells of three strains of Dictyosrrlium discoidruin , NC-4. γs-13 and γs-18. for killing. fruiting body formation, spore formation and viability of the spores were studied.
The strain of γs-13 was more sensitive to UV light for killing than NC-4 at 10% survival. In addition. γs-13 was the most sensitive strain among the three for fruiting body formation and spore formation. The developmental process of this organism, however, showed a large resistance to UV light when compared with the killing. The spores of γs-13 formed after UV irradiation were mostly non-viable, though those of γs-18 and NC-4 were fully viable     

DNA REPAIR IN ULTRAVIOLET LIGHT IRRADIATED HeLa CELLS AND ITS REVERSIBLE INHIBITION BY HYDROXYUREA

Abstract— –The repair of u.v. damaged DNA in HeLa cells can be detected using the alkaline sucrose gradient technique. As a result of pyrimidine dimer excision single strand breaks are produced in DNA of irradiated cells. Rejoining of these breaks occurs during an 8 hr post-irradiation incubation period and is prevented by hydroxyurea and acriflavine. The inhibition of repair by hydroxyurea can be reversed by a mixture of all 4 deoxyribonucleosides at a concentration that does not reverse the inhibition of total DNA synthesis.     

ENHANCEMENT OF ULTRAVIOLET OR N-METHYL-N'- NITRO-N-NITROSOGUANIDINE SENSITIVITY OF DICTYOSTELIUM DISCOIDEUM BY 3-AMINOBENZAMIDE

Abstract— The amoeboid cells of Dktyostelium discoideum NC–4 possess a 3-aminobenzamide(3-ABA)-sensitive repair mechanism for DNA damages induced by UV-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treatment. We have studied the effect of 3-ABA on each step of excision repair in the UV- irradiated cells. Although the nicking of DNA-strand and the excision of pyrimidine dimcrs are insensitive to 3-ABA, the rejoining of DNA strand-breaks is sensitive. The frequency of mutation induced by UV-irradiation or MNNG-treatment is depressed by 3-ABA. The mechanisms of repair inhibition by 3-ABA are discussed.     

SURVIVAL, SPORE FORMATION AND EXCISION REPAIR OF UV-IRRADIATED DEVELOPING CELLS OF DICTYOSTELIUM DISCOIDEUM NC-4

Abstract— The synchronously developing aggregates of the cellular slime mold, Dictyostelium discoideum NC-4, were disaggregated into individual cells and irradiated with 254 nm UV light at preaggregation (0h), late interphase (6h), late aggregation (12 h), and preculmination (18 h). When assayed for replica-tive ability (colony formation), the developing cells at 0, 6, 12, and 18h showed the same sensitivity as vegetative cells; the 10% survival dose (D₁₀) was 160 J/m². The spores were more sensitive, with D₁₀ of 70 J/m². Excision repair of the nuclear DNA of the developing cells was studied by alkaline sucrose gradients. UV-induced single-strand breakage and rejoining of the DNA occurred to the same final extent in the cells from the 0, 6, 12 and 18 h stages of development, but a longer time was required for the completion of rejoining at the later stages (for example, at 54 J/m², 6.6 h for preculmination cells, 3.3 h for preaggregation cells). When the cells irradiated at various stages were required to redevelop, as measured by the relative numbers of spores produced, their sensitivity for completing this development increased the later the stage from which they were taken. The D₁₀s for spore production were 200, 130, 100 and 70 J/m² for cells at the 0, 6, 12 and 18 h stages, respectively. The fractional viability among the spores that appeared after this treatment was the same independent of the stage at which the cells were irradiated; the D₁₀ for this viability was 160 J/m², the same as if the cells had been plated immediately with no intervening developmental sequence. We conclude that DNA excision repair as related to replicative ability is retained at all stages of development; however, development seems independent of replicative ability and depends upon DNA and/or non-DNA damage in a more complex way.     

TOXICITY, DNA DAMAGE AND INHIBITION OF DNA REPAIR SYNTHESIS IN HUMAN MELANOMA CELLS BY CONCENTRATED SUNLIGHT

A 1 m diameter water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV, in that D _q, was usually larger than D _o. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line used for further studies, DNA strand breaks and DNA-protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers (paper chromatography) and DNA interstrand crosslinking (alkaline elution) could not be detected. The solar fiuence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D _o value and then declining; semiconservative DNA synthesis on the other hand remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis.     

PSORALEN PLUS ULTRAVIOLET RADIATION-INDUCED INHIBITION OF DNA SYNTHESIS AND VIABILITY IN HUMAN LYMPHOID CELLS IN VITRO*

Abstract— 8-Methoxypsoralen (8-MOP) plus high intensity long wavelength ultraviolet radiation (UV-A) is presently being used to induce remissions of psoriasis and mycosis fungoides. Previous studies demonstrated inhibition of DNA synthesis in circulating leukocytes from some patients during this therapy. The present study is designed to determine whether conditions of 8-MOP concentration and UV-A exposure attained during therapy might be sufficient to result directly in decreased lymphoid cell DNA synthesis and viability in vitro. Tritiated thymidine (³HTdR) incorporation and cell growth in suspension culture following UV-A exposure alone or with therapeutic concentrations of 8-MOP was examined in peripheral blood lymphocytes and in Ebstein-Barr virus transformed human lymphoblas-toid cell lines. UV-A exposure alone induced a dose-dependent inhibition of HTdR incorporation in both types of lymphoid cells (3000 J/m² resulted in 77% of control ³HTdR incorporation). Pre-incubation with 0.1 μg/m/ 8-MOP before UV-A exposure induced a significantly greater inhibition of ³HTdR incorporation (3000 J/m² resulted in 61% of control ³HTdR incorporation). Greater inhibition of ³HTdR incorporation was observed by preincubation of the lymphoblastoid cells with 1.0μg/mC 8-MOP (3000 J/m² resulted in 41% of control) but not in the lymphocytes (3000 J/m² resulted in 63% of control). The concentration of viable lymphoblastoid cells did not decrease below the original concentration after the highest dose of UV-A alone (29,000 J/m²) but preincubation with 0.1 μg/mC 8-MOP resulted in 40% survival after 3000 J/m² (D₃₇ approximately 3000 J/m²) and preincubation with 1.0 μg/ 8-MOP resulted in 0.6% survival after 3000 J/,² (D₃₇ approximately 800 J/m²). This study suggests that the low doses of 8-MOP and UV-A received by patients' lymphocytes may be sufficient to explain the decreased DNA synthesis found in their circulating leucocytes. The long term consequences of such damage remain to be determined.     

THIOPYRONINE- AND 8-METHOXYPSORALEN-SENSITIZED PHOTODYNAMIC EFFECT ON CELL GROWTH, COLONY FORMING ABILITY and RNA SYNTHESIS IN Saccharomyces MUTANTS DEFICIENT IN DNA REPAIR

Abstract –We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8-methoxypsoralen (8-MOP) and ultraviolet A (UV-A) light, on growth, colony forming ability and RNA synthesis in a repair-proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8-MOP and UV-A repair-deficient mutants were significantly more sensitive than the repair-proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.     

POSTREPLICATION REPAIR IN AN EXCISION-DEFECTIVE MUTANT OF ESCHERICHIA COLI: ULTRAVIOLET LIGHT-INDUCED INCORPORATION OF BROMODEOXYURIDINE INTO PARENTAL DNA*

Abstract— Ultraviolet (UV) light-induced incorporation of bromodeoxyuridine (BrdUrd) into parental DNA of an excision-defective mutant of Escherichia coli has been observed by selective photolysis of bromouracil (BrUra)-containing regions in the parental DNA. It appears that the BrUra-containing regions occur only in that DNA which has served as a template for normal semiconservative replication. After an exposure at 254 nm which results in one pyrimidine dimer per 45times 10⁶ daltons, incubation in BrdUrd resulted in BrUra–containing regions ˜ 1.5 times 10⁴ nucleotides in length at intervals of ˜ 55 times 10⁶ daltons in the parental DNA. Thus approximately one BrUra-containing region has occurred for every 1.2 pyrimidine dimers in the parental DNA. The observed incorporation of BrdUrd is interpreted in terms of a proposed model for postreplication repair in which genetic exchanges produce single-strand gaps in the parental DNA.     

THE NATURE OF ULTRAVIOLET LIGHT-INDUCED STRAND BREAKAGE IN DNA CONTAINING BROMOURACIL

Abstract— Single-strand breaks are produced in the phosphodiester backbone of ultraviolet-light-irradiated 5–bromodeoxyuridine-containing DNA (BU-DNA) after treatment with alkali. No radiation dependent breakage is observed in thymine-containing DNA (thy-DNA). The relative yields of breaks terminated by 5'-hydroxyl and 5'-phosphate groups was determined by measuring the rate of phosphorylation achieved with polynucleotide kinase in BU-DNA single strands before and after treatment with alkaline phosphatase. The ratio of 5'-phosphate to 5' hydroxyl groups ranged from 2.3 to 2.9 in different experiments. When cysteamine was present during irradiation no new end groups were produced.
In order to identify the nucleoside(s) at the 5'-termini, phosphate groups were removed with alkaline phosphatase and the 5'-hydroxyl groups were phosphorylated with polynucleotide kinase. Electrophoresis of enzymatic digests showed a single ³²P-labeled component migrating more rapidly than any of the four usual 5'-mononucleotides. Upon column chromatography this component resolved into a major peak coincident with 5'-dUMP and a lesser unidentified constituent. No 5'-dBUM³²P was observed among these nucleotides.     

VISUALIZATION OF ULTRAVIOLET LIGHT-INDUCED THYMINE DIMERS IN DNA BY IMMUNOELECTRON MICROSCOPY

Abstract— To see the damage of DNA due to ultravoilet-B more distinctly, immunoelectron microscopic studies using a monoclonal antibody against cyclobutane-type thymine dimers were performed. As a result, we could detect the existence of thymine dimers on human genomic DNA and _pUC18 plasmid DNA visually. This technique can be useful to locate the photoproducts formed on DNA.     

RESEARCH NOTE: THIOPYRONINE-AND 8-METHOXYPSORALEN-SENSITIZED PHOTODYNAMIC EFFECT ON CELL GROWTH, COLONY FORMING ABILITY AND RNA SYNTHESIS IN Saccharomyces MUTANTS DEFICIENT IN DNA REPAIR

Abstract: We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8-methoxypsoralen (8-MOP) and ultraviolet A (UV-A) light, on growth, colony forming ability and RNA synthesis in a repair-proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8-MOP and UV-A repair-deficient mutants were significantly more sensitive than the repair-proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.     

FORMATION OF NON-VIABLE SPORES OF DICTYOSTELIUM DISCOIDEUM BY UV-IRRADIATION AND CAFFEINE

Abstract We have characterized the spores formed from amoeboid cells of the wild type strain of Dictyostelium discoideum after UV-irradiation. Cell differentiation in the presence of caffeine after a fluence of 300 J/m² resulted in a population of spores which was 98% non-viable. The UV-irradiation did not affect the conversion of the spores to swollen spores but did affect the conversion of swollen spores to amoeboid cells. When the germination of the spores was done without caffeine, we detected only a small effect on conversion of swollen spores to amoeboid cells and on the beginning of growth. On the other hand, in the presence of caffeine, the spores had a remarkable delay in both. It was also shown that few, if any, pyrimidine dimers exist in the DNA of the non-viable spores. Possible mechanisms of formation of non-viable spores are discussed.     

INHIBITION OF PROTEIN SYNTHESIS IN CULTURED TOBACCO CELLS BY ULTRAVIOLET RADIATION

Abstract— Ultraviolet (254 nm) irradiation of liquid-cultured tobacco cells strongly and quickly inhibited their ability to incorporate labeled amino acids into protein. An incident dose of only 388 J/m² reduced incorporation to 37 per cent of the original rate. The effect on amino acid incorporation did not seem to depend on inhibition of amino acid uptake, inhibition of the supply of nucleoside triphosphates, or inhibition of the supply of messenger RNA to cytoplasmic ribosomes.     

VIABILITY OF THE SPORES FORMED AFTER UV IRRADIATION IN DICTYOSTELIUM DISCOIDEUM

Both abilities of germination of spores formed after UV irradiation and of growth of amoeboid cells emerged from the spores were studied on two kinds of Dictyostelium discoideum strains, NC-4 and ys-13.
An inhibition of germination was observed on the spores of ys -13 when formed after UV irradiation, while no inhibition was detected on the ability of germination of spores of NC-4. The amoeboid cells of ys -13 emerged from the spores showed a heavy delay of growth, although no delay of growth was detected even on the amoeboid cells of NC-4 emerged from the spores formed after UV irradiation. The strain of NC-4 must repair UV lesions fully before spore formation, while the spores of ys-13 must keep some UV lesions unrepaired and send them to the next generation of amoeboid cells. The characters of UV lesion inheritable through the spores to the next amoeboid cells in ys-13 were discussed.