Synthesis of Deoxyribonucleic Acid After Ultraviolet Irradiation of Sensitive and Resistant Haemophilus influenzae

Synthesis of deoxyribonucleic acid (DNA) has been measured as a function of ultraviolet (UV) radiation dose in wild-type and seven UV-sensitive strains of Haemophilus influenzae. At the UV doses used, all strains were able to resume DNA synthesis, even those which are unable to excise pyrimidine dimers from their DNA. These excisionless strains showed longer UV-induced delays in DNA synthesis than all but one of the other strains. The longest delay was shown by DB117, a strain which can excise dimers but which is recombination deficient and unable to rejoin X ray-induced single-strand breaks. All strains showed a progressive decrease in sensitivity as they approached the stationary phase.     

Effect of host lex, recA, recF, and uvrD genotypes on the ultraviolet light-protecting and related properties of plasmid R46 in Escherichia coli.

The ability of plasmid R46 to reduce the lethal but enhance the mutagenic effect of ultraviolet (UV) irradiation was tested in sets of Escherichia coli K-12 derivatives, wild type or with different mutations affecting DNA repair capacity, but otherwise isogenic. UV protection and enhancement of UV mutagenic effect were obtained in uvrA6, uvrB5, uvrD3, and recF143 hosts, but not in a recA56 strain. The plasmid gave some UV protection in two lexA1 and two lexA101 strains and in one lexA102 host, but produced no such effect in another lexA102 host. The plasmid restored UV mutagenic effect in a lexB30 strain, the yield of induced mutants per survivor of irradiation (10 J/m2) being about the same for the lexB30(R46) and lex+(R46) strains; by contrast the plasmid, though it reduced the UV sensitivity of the lexB30 strain, did not make it as UV-resistant as the lex+ R-strain.     

Inactivation of carotenoid-producing and albino strains of Neurospora crassa by visible light, blacklight, and ultraviolet radiation.

Suspensions of Neurospora crassa conidia were inactivated by blacklight (BL) radiation (300 to 425 nm) in the absence of exogenous photosensitizing compounds. Carotenoid-containing wild-type conidia were less sensitive to BL radiation than albino conidia, showing a dose enhancement factor (DEF) of 1.2 for dose levels resulting in less than 10% survival. The same strains were about equally sensitive to shortwave ultraviolet (UV) inactivation. The kinetics of BL inactivation are similar to those of photodynamic inactivation by visible light in the presence of a photosensitizing dye (methylene blue). Only limited inactivation by visible light in the absence of exogenous photosensitizers was observed. BL and UV inactivations are probably caused by different mechanisms since wild-type conidia are only slightly more resistant to BL radiation (DEF = 1.2 at 1.0% survival) than are conidia from a UV-sensitive strain (upr-1, uvs-3). The BL-induced lethal lesions are probably no cyclobutyl pyrimidine dimers since BL-inactivated Haemophilus influenzae transforming deoxyribonucleic acid is not photoreactivated by N. crassa wild-type enzyme extracts, whereas UV-inactivated transforming deoxyribonucleic acid is photoreactivable with this treatment.     

The effect of post-UV dark incubation on survival of chick-embryo fibroblasts after photoreactivation

A survival assay with chick-embryo fibroblasts was used to study photoreactivation of ultraviolet (UV) irradiation-induced damage. The kinetics of the photoreactivation was studied as a function of the length of a post UV dark incubation period of from 0 to 18 h at 38.5 degrees C. The logarithmic survival curve with no photoreactivation had a Do of about 4.3 J/m2 giving approximately 0.8% relative plating efficiency after a UV dose of 21 J/m2. At this dose the efficiency of photoreactivation (survival increase per unit blacklight dose) increased with post UV incubation time reaching a maximum at 4-6 h, then declining until there was little photoreactivation observed for times longer than about 11 h. The possibility that this effect was produced by pre-UV perturbations of the cell cycle was eliminated by the fact that the same results were achieved after several rather different trypsinization protocols. The shape of the photoreactivation vs. blacklight curve at the time of peak efficiency showed a threshold up to about 3 kJ/m2, a rising portion and a plateau after 12-16 kJ/m2 when the survival increased by a factor of roughly 8.     

Studies on the ultraviolet light sensitivity of Bloom's syndrome fibroblasts

The sensitivity of Bloom's syndrome (bl/bl) fibroblasts to ultraviolet light (254 nm) has been estimated by 4 criteria: sister-chromatid exchange (SCE) formation, micronucleus production, cell survival, and host-cell reactivation of UV-irradiated adenovirus 2. In general, bl/bl strains did not differ significantly from the normal (+/+) strains in their response to UV treatment by any of the 4 criteria. One bl/bl strain, GM1492, was exceptional: It was abnormally sensitive to UV light in the SCE, micronucleus, and host-cell reactivation assays, but was not sensitive to UV as estimated by colony-forming ability. Thus, although one of the bl/bl strains studied in the experiments was sensitive to UV light as judged by some criteria, UV sensitivity is not a universal characteristic of Bloom's syndrome cells. It is nuclear whether the UV sensitivity of the GM1492 strain reflects genetic diversity within the syndrome or some unrelated property of this strain.     

Repair of Deoxyribonucleic Acid in Haemophilus influenzae I. X-Ray Sensitivity of Ultraviolet-sensitive Mutants and Their Behavior as Hosts to Ultraviolet-irradiated Bacteriophage and Transforming Deoxyribonucleic Acid

Seven mutants of Haemophilus influenzae were isolated by the criterion of sensitivity to ultraviolet (UV) inactivation of colony formation. These mutants and the wild type were characterized with regard to X-ray inactivation of colony formation, UV induction of division inhibition, the ability of the eight strains to act as recipients to UV-irradiated H. influenzae phage and transforming deoxyribonucleic acid (DNA), and the influence of acriflavine on the survival of UV-irradiated transforming DNA with these strains as recipients. The photoreactivable sector of transforming DNA with yeast photoreactivating enzyme was measured for the most UV-sensitive mutant and was found to be greater than that of wild type. Judged by the above criteria, the order of the strains' sensitivities shows some, but by no means complete, correlation from one type of sensitivity characterization to another, indicating that a minimum of two variables is needed to explain the differences in the strains. Acriflavine increases the UV sensitivity of transforming DNA except in the most sensitive mutant. This effect is usually, but not always, more pronounced in the case of the more UV-resistant marker. The acriflavine effect is postulated to be the result of at least two factors: (i) interference with repair of transforming DNA in the host cell, and (ii) interference with the probability of recombination between transforming DNA and host DNA.     

Repair of Ultraviolet Radiation Damage in Sensitive Mutants of Micrococcus radiodurans

Various aspects of the repair of ultraviolet (UV) radiation-induced damage were compared in wild-type Micrococcus radiodurans and two UV-sensitive mutants. Unlike the wild type, the mutants are more sensitive to radiation at 265 nm than at 280 nm. The delay in deoxyribonucleic acid (DNA) synthesis following exposure to UV is about seven times as long in the mutants as in the wild type. All three strains excise UV-induced pyrimidine dimers from their DNA, although the rate at which cytosine-thymine dimers are excised is slower in the mutants. The three strains also mend the single-strand breaks that appear in the irradiated DNA as a result of dimer excision, although the process is less efficient in the mutants. It is suggested that the increased sensitivity of the mutants to UV radiation may be caused by a partial defect in the second step of dimer excision.     

Transitory germinative excision repair in Bacillus subtilis.

Bacillus subtilis strains UVSSP-42-1 (hcr42 ssp1) and UVSSP-1-1 (hcr1 ssp1) are ultraviolet (UV) radiation sensitive both as dormant spores and as vegetative cells. These strains are unable to excise cyclobutane-type dimers from the deoxyribonucleic acid (DNA) of irradiated vegetative cells and fail to remove spore photoproduct from the DNA of irradiated spores either by excision (controlled by gene hcr) or by spore repair (controlled by gene ssp1). When irradiated soon after spore germination, these strains excise dimers, but not spore photoproduct, from their DNA. This process, termed germinative excision repair, functions only transiently in the germination phase and is responsible for the high UV resistance of germinated spores and for their temporary capacity to host cell reactivate irradiated phages infecting them. The recA1 mutation confers higher UV sensitivity to the germinated spores, but does not interfere with dimer removal by germinative excision repair.     

Phototoxic effect of UVR on wild type, ebony and yellow mutants of Drosophila melanogaster : Life Span, fertility, courtship and biochemical aspects

Melanin plays an important role in protecting organisms from ultraviolet radiation (UVR). Therefore, it is possible that differently colored strains can show different sensitivities to UVR. In the present work, life span, fertility and courtship behavior of wild type (w), ebony (e) and yellow (y) strains of Drosophila melanogaster were studied to evaluate their sensitivity to ultraviolet (UV). Because a range of photo- toxic effects of UVR are mediated through generation of free radicals, levels of free radicals, lipid per- oxide (malondialdehyde, MDA) and superoxide dismutase (SOD) activity of three strains were examined to indicate their antioxidant defending ability and oxidative status. It was shown that w always had the highest lifespan and fertility not only in the control but also in UV-exposed groups. Moreover, lifespan and fertility of e were significantly higher (P<0.0001) than those of y in the UV-exposed groups, but not for the control. On the other hand, UV exposure had an adverse effect on courtship of flies. Stronger electron paramagnetic resonance (EPR) signals could be detected in w, e and y exposed to 5 min UV. And there were more significant changes of EPR signals in y than in w and e. UVR had no significant (P=0.1782) effect on the SOD activities. After pooling data from the control and UV-exposed groups, we found that w had a significantly (P<0.05) higher level of SOD activity, but e and y were nearly at the same levels (P>0.05). MDA levels were increased in the UV dose-dependent manner (P=0.0495). In con- clusion, our results suggested that UVR can decrease life span and fertility of flies and do harm to courtship, which may be due to oxidative damage to flies tissues (e.g. central nervous system) induced by free radicals. w had the highest tolerance to UVR, which may be ascribed to its advantage of survival under the natural condition and at high level of SOD activity. Then differences of pigment between e and y in absorbing UV, shielding against UV and scavenging free radicals produced by UVR should be responsible for their different sensitivity to UVR.     

Deoxyribonucleic acid repair in Escherichia coli mutants deficient in the 5''----3'' exonuclease activity of deoxyribonucleic acid polymerase I and exonuclease VII.

A series of Escherichia coli strains deficient in the 5'----3' exonuclease activity associated with deoxyribonucleic acid (DNA) polymerase I (exonuclease VI) and exonuclease VII has been constructed. Both of these enzymes are capable of pyrimidine dimer excision in vitro. These strains were examined for conditional lethality, sensitivity to ultraviolet (UV) and X-irradiation, postirradiation DNA degradation, and ability to excise pyrimidine dimers. It was found that strains deficient in both exonuclease VI (polAex-) and exonuclease VII (xseA-) are significantly reduced in their ability to survive incubation at elevated temperature (43 degrees C) beyond the reduction previously observed for the polAex single mutants. The UV and X-ray sensitivity of the exonuclease VI-deficient strains was not increased by the addition of the xseA7 mutation. Mutants deficient in both enzymes are about as efficient as wild-type strains at excising dimers produced by up to 40 J/m2 UV. At higher doses strains containing only polAex- mutations show reduced ability to excise dimers; however, the interpretation of dimer excision data at these doses is complicated by extreme postirradiation DNA degradation in these strains. The additional deficiency in the polAex xseA7 double-mutant strains has no significant effect on either postirradiation DNA degradation or the apparent deficiency in dimer excision at high UV doses observed in polAex single mutants.     

Mutants of Yeast Sensitive to Ultraviolet Light

Six uvr mutants of Saccharomyces cerevisiae with hypersensitivity to ultraviolet (UV) light were isolated after mutagen treatment with ethylmethanesulfonate. UV sensitivity ranges from moderate to extreme, and four of the mutants are also sensitive to nitrous acid. Ranking in terms of UV sensitivity does not parallel ranking in terms of nitrous acid sensitivity. Homozygous diploid mutant strains are somewhat less sensitive than the corresponding haploids. All mutations are recessive. None of the mutants is sensitive to gamma rays, and each shows photoreactivation after UV radiation. Complementation tests and tetrad analysis indicate that each strain represents mutation in a different gene. Two of the uvr genes are linked, and two others are centromere-linked.     

Genetically controlled removal of "spore photoproduct" from deoxyribonucleic acid of ultraviolet-irradiated Bacillus subtilis spores

Previous genetic analysis indicated that at least two genes determine the ultraviolet (UV) sensitivity of Bacillus subtilis spores. The present study shows that these genes independently control two distinguishable processes for removing UV-induced spore photoproduct (5-thyminyl-5,6-dihydrothymine, or TDHT) from spore deoxyribonucleic acid. The first, is a spore repair mechanism by which TDHT is removed rapidly without appearing in acid-soluble form. This mechanism, which is demonstrated in both UV-resistant and excision-deficient strains, operates to a certain extent during germination without requiring vegetative growth. The second, demonstrated in a mutant which lacks the first mechanism, removes TDHT relatively slowly and only if germinated spores are allowed to develop toward vegetative cells. The latter mechanism appears identical to excision-resynthesis repair, since the mutation abolishing it renders the irradiated vegetative cells incapable of removing cyclobutane-type pyrimidine dimers. Blocking either one of these mechanisms only slightly affects the UV sensitivity of spores, but blocking both prevents TDHT removal and gives high UV sensitivity.     

Dominant Mutations (lex) in Escherichia coli K-12 Which Affect Radiation Sensitivity and Frequency of Ultraviolet Light-Induced Mutations

Three mutations, denoted lex-1, -2 and -3, which increase the sensitivity of Escherichia coli K-12 to ultraviolet light (UV) and ionizing radiation, have been found by three-factor transduction crosses to be closely linked to uvrA on the E. coli K-12 linkage map. Strains bearing these mutations do not appear to be defective in genetic recombination although in some conjugational crosses they may fail to produce a normal yield of genetic recombinants depending upon the time of mating and the marker selected. The mutagenic activity of UV is decreased in the mutant strains. After irradiation with UV, cultures of the strains degrade their deoxyribonucleic acid at a high rate, similar to recA(-) mutant strains. Stable lex(+)/lec(-) heterozygotes are found to have the mutant radiation-sensitive phenotype of haploid lex(-) strains.     

Escherichia coli ras locus: its involvement in radiation repair

There are several classes of Escherichia coli mutants defective in radiation repair. These include strains defective in pyrimidine dimer excision, in photoreactivation, in recombination, in repair of X-ray damage, and ultraviolet (UV)-conditional mutants which do not divide after UV. Another mutant (ras(-)) has been isolated. The ras(-) has increased UV sensitivity, but only slightly increased X-ray sensitivity (1.5-fold increase). Ability to effect genetic recombination, to reactivate irradiated bacteriophage T1, and to be photoreactivated is normal. UV-induced mutation frequency is greatly increased in the mutant. The ras(-) apparently lacks the ability to repair some UV damage in the bacterial cell but can repair UV damage to bacteriophage DNA. The ras locus is located between lac and purE on the chromosome map.     

The shape of the blacklight dose photoreactivation curve for chick embryo fibroblasts

The curves of UV (254 nm) induced pyrimidine dimers (endonuclease sensitive sites) vs. photoreactivating blacklight (365 nm) dose for cultured chick embryo fibroblasts reveal several new features. When the cells are incubated in the dark at 37 degrees following UV (254 nm) treatment, the efficiency of subsequent photorepair increases for the first few hours post-UV. The efficiency then remains approximately constant for several hours. Photorepair data obtained during this later period were plotted as the logarithm of dimer-enzyme complexes available for photoreactivation vs. blacklight (365 nm) dose. For a fixed damaging UV (254 nm) dose, the resulting curve has a shoulder of approximately 6-10 kJ/m2 followed by a straight line portion with a slope of magnitude about 1.5 X 10(-4) m2/J for UV doses up to 15 J/m2. For higher UV doses the shoulder remains about the same, but the slope decreases in magnitude. The shoulder is interpreted to indicate that a light-dependent step is necessary to activate the enzyme. The decrease in slope with increased UV dose together with some split photoreactivation dose experiments suggests that some site-to-site motion and multiple site function of the photorepair enzyme molecules may come into play at the higher levels of damage, but the evidence indicates that these complications are relatively unimportant at low UV doses.     

Dominance of Ultraviolet Radiation Resistance in Partial Diploids of Escherichia coli K-12

Although an F'13 capR(+)/capR9 strain is nonmucoid and an F'13 capR9/capR(+) strain is mucoid, both strains are ultraviolet (UV)-resistant. In contrast, haploid capR9 strains are UV-sensitive. Therefore, UV resistance is dominant to UV sensitivity, regardless of whether the capR(+) allele is on the chromosome or on the F'13 episome.     

Abnormal sensitivity of some Cockayne's syndrome cell strains to UV- and gamma-rays. Association with a reduced ability to repair potentially lethal damage

Cockayne's syndrome (CS) is a rare autosomal recessive genetic disease characterized by mental and physical retardation, microcephaly, dwarfism, retinitis pigmentosa and a hypersensitivity to sunlight. Cells originating from patients also exhibit, in vitro, a hypersensitivity to UV radiation. Using a colony assay in vitro, we studied the sensitivity of 5 CS cell strains (GM739, BOR, CS697, CS698 and KA) and two normal ones (HF19 and GP) to UV- and gamma-irradiation. The 5 CS strains appear to be UV-hypersensitive but the sensitivity varies widely from one strain to another. Hypersensitivity to gamma-rays has been reported for 4 out of the 5 CS cell strains investigated. However, these CS cell strains are less sensitive to gamma-rays than are ataxia telangiectasia cells. The KA cell strain exhibits a normal response to gamma-irradiation. Repair of potentially lethal damage (PLD) after UV- and gamma-irradiation was investigated by using unfed plateau-cell cultures. Under these conditions, control cells show a great capacity to repair PLD (10- to 30-fold survival increase at 1% survival level). The two CS strains (GM739 and BOR), which are hypersensitive to both UV- and gamma-irradiation, exhibit no or only little PLD repair after treatment. In contrast, the normal response of KA cells to gamma-rays is associated with a normal PLD repair capability. This latter cell strain exhibits an intermediate sensitivity to UV and shows an intermediate PLD repair capacity. The response of CS cell strains after gamma-irradiation suggests a genetic heterogeneity. Three complementation groups are described in CS cells when dealing with UV radiosensitivity. However, variations in gamma-ray sensitivity are reported for cells within the same UV complementation group.     

Isolation, Characterization, and Genetic Analysis of Mutator Genes in Escherichia coli B and K-12

Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut(+)) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC. In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA(+) transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator properly and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC.     

Induction and Characterization of Chlorate-resistant Strains of Rosa damascena Cultured Cells

The sensitivity of Rosa damascena cultured cells to chlorate was measured by plating samples of suspensions in agar containing NaClO₃. This sensitivity depended on the age of the cultures that were plated. Chlorate-resistant colonies isolated from 5- to 7-day cultures retained their resistance through many generations of growth in medium lacking NaClO₃; they also retained resistance when mixed with sensitive cells. Treating cell aggregates with ultraviolet (UV) light (254 nanometers), or UV light (360 nanometers) in the presence of 4′-methoxymethyltrioxsalen, increased the proportion that was resistant to NaClO₃. However, the amount of increase was low (three times) and required very specific doses of UV light. The UV treatments did not select for chlorate-resistant cells over chlorate-sensitive cells. The data suggested that UV had induced mutations leading to chlorate resistance. Approximately 15% of the resistant strains did not grow on medium containing nitrate as the sole nitrogen source. These strains lacked ability to reduce chlorate to chlorite. This observation supports the current idea that chlorate toxicity depends on the activity of nitrate reductase. Approximately 85% of the resistant strains grew on medium containing nitrate as the sole nitrogen source. These strains lost catalase activity following chlorate treatment, indicating that they took up and reduced chlorate. These strains have a mechanism for tolerating chlorate and its reduction products, rather than avoiding them.     

Interaction of the exr and lon Genes in Escherichia coli

Strains of Escherichia coli carrying the gene lon typically produced excess capsular polysaccharide, and were sensitive to ultraviolet light (UV) irradiation, thymine starvation, and nalidixic acid, forming long filaments after these treatments. Sensitivity was reduced by a number of posttreatments. In the presence of a second UV sensitivity gene, exr, some of these properties were suppressed: long filaments were not formed, the effect of lon on UV and nalidixic acid sensitivity was greatly reduced, and irradiation posttreatments gave an enhancement of survival characteristic of exr rather than lon strains. Production of capsular polysaccharide was not affected by the exr gene.