Ultraviolet mutagenesis of radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans

A mutational tester strain (JP10) of the nematode C. elegans was used to capture recessive lethal mutations in a balanced 300 essential gene autosomal region. The probability of converting a radiation interaction into a lethal mutation was measured in young gravid adults after exposure to fluences of 254-nm ultraviolet radiation (UV) ranging from 0 to 300 Jm-2. Mutation frequencies as high as 5% were observed. In addition, three different radiation-hypersensitive mutations, rad-1, rad-3 and rad-7 were incorporated into the JP10 background genotype, which allowed us to measure mutation frequencies in radiation-sensitive animals. The strain homozygous for rad-3 was hypermutable to UV while strains homozygous for rad-1 and rad-7 were hypomutable. Data showing the effects of UV on larval development and fertility for the rad mutants is also shown and compared for wild-type and JP10 backgrounds.     

Characteristics of DNA repair of a UV-sensitive mutant (radC) of Dictyostelium discoideum

Summary A radiation-sensitive mutant, TW8(radC), of Dictyostelium discoideum is more sensitive to ultraviolet light (UV) killing than the parental wild strain NC4(RAD ⁺), but is resistant to 4-nitroquinoline 1-oxide (4NQO) at almost the same level as NC4. In TW8 amoebae, single-strand breaks of DNA molecules were hardly detectable immediately after UV irradiation, and the removal of pyrimidine dimers was depressed during the postirradiation incubation when compared with that of NC4 amoebae. After treatment with 4NQO, however, single-strand breaks were detected in TW8 amoebae. The almost complete rejoining of these breaks was also detected after the removal of 4HAQO-adducts. The TW8 amoebae have an efficient repair capacity against DNA damage caused by 4NQO, MMS, MMC and MNNG but not UV.Abbreviations 4NQO 4-nitroquinoline 1-oxide - MMS methyl methanesulphonate - MMC mitomycin C - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Effects of age and liquid holding on the UV-radiation sensitivities of wild-type and mutant Caenorhabditis elegans dauer larvae

The dauer larva is a facultative developmental stage in the life cycle of the nematode Caenorhabditis elegans. Dauer larvae, which can survive under starvation for over 60 days, resume normal development when feeding is resumed. Wild-type (N2) and 4 radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV-irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, rad-2 and rad-7 mutants were not hypersensitive. Irradiation also delayed development in survivors. Wild-type dauer larvae did not differ in radiation sensitivity from 0 through 50 days of age. There was no liquid holding recovery (LHR); that is, survival did not increase when wild-type dauer larvae were held in buffer after irradiation.     

Allele specific and locus non-specific suppressors in Aspergillus nidulans.

Using N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, ethyl methanesulphonate or 4-nitroquinoline-1-oxide mutagenesis and an enrichment method for the isolation of auxotrophs, 25 mutants with defects in the adA locus were obtained after screening 41,376 colonies. One of these, adA24, did not complement with any of the other adA mutants, had a very high reversion rate and had some other properties which usually characterize strains carrying nonsense mutations. All revertants of adA24 carried dominant suppressor mutations. A group of adA24 suppressors was tested for allele and locus specificity. They were found to suppress only some adA alleles, and at the same time, some mutations in the methG, methH, argB and proA loci. It is proposed that the allele specific and locus non-specific adenine suppressors are suppressors of nonsense mutations.     

Caffeine enhancement of radiation killing in different strains of Saccharomyces cerevisiae.

Summary Haploid and diploid wild type strains, and three classes of radiation-sensitive mutants of Saccharomyces cerevisiae were tested for enhancement of UV-inactivation by caffeine in growth medium. In addition, the sensitizing effect of caffeine was studied in a haploid and a diploid wild type strain after gamma-irradiation. The drug sensitized the UV-irradiated cells of all strains except those reported to be only slightly UV-sensitive but highly sensitive to ionizing radiation. After gamma-irradiation, no caffeine-enhancement of killing was observed in stationary phase cells of either the haploid or the diploid strain. However, log-phase cells of both strains were partially sensitized.The results of both sets of experiments suggested that caffeine interferes with a recombinational repair occurring in cells in S or G2 phase.     

Post-treatment with caffeine and the induction of gene mutations by ultraviolet irradiation and ethyl methanesulphonate in V-79 Chinese hamster cells in culture.

The effect of caffeine on V-79 Chinese hamster cells after ultraviolet irradiation or treated with ethyl methanesulphonate was investigated. Caffeine strongly potentiated the killing of both agents, but it had no effect on the induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. The results are consistent with the notion that caffeine slows down an error-prone post-replicative repair mechanism without changing the mutation frequency.     

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.     

Identification and characterization of catA, a mutation causing catalase deficiency in Dictyostelium discoideum.

Various strains of Dictyostelium discoideum were assayed for catalase activity. We were able to demonstrate the presence of catalase in lysates of all strains tested except one, ts12m. Lysates of this strain did not show any detectable level of catalase. The increased sensitivity of intact ts12m amoebae to hydrogen peroxide was consistent with the catalase deficiency. We followed catalase activity through a developmental time course for the wild type (Ddb) and were able to show its presence throughout development. No catalase activity was detected from ts12m in any stage of development. The growth and development of this acatalasemic strain of D. discoideum was no different than those of the wild type. The mutation, catA, was assigned to linkage group II.     

Specificity and frequency of ultraviolet-induced reversion of an iso-1-cytochrome c ochre mutant in radiation-sensitive strains of yeast

The basis for the specific pattern of ultraviolet-induced reversion of cyc1-9, an ochre allele of the structural gene for iso-1-cytochrome c, has been examined in radiation-sensitive strains of yeast. Previous analysis, using RAD⁺ strains, showed that 21 out of 23 cyc1-9 revertants induced by ultraviolet light arose by A · T to G · C transition at the first position in the UAA codon, the remaining two occurring by A · T to T · A transversion at the second position (Stewart et al., 1972; Sherman &; Stewart, 1974). All possible base-pair substitutions could be obtained with the aid of other mutagens.It has now been shown that this specificity depends largely on the action of the RAD6 locus, since ultraviolet-induced revertants of cyc1-9 arose by a variety of base-pair substitutions in a strain carrying the rad6-1 allele. Induced reversion frequencies in strains carrying this allele are much lower than normal, though significantly higher than the spontaneous frequency, and the strains are more sensitive to the lethal effects of both ultraviolet and X-irradiation. The phenotypically similar rad18-2 mutation, which appears to block the same repair pathway as rad6-1, also has some effect on the reversion specificity, but its action depends on the presence of other, unidentified, mutations. Specificity was, however, completely unaltered in an excision-defective strain carrying the rad1-2 allele. Induced reversion frequency of cyc1-9 was much higher than normal in this strain. Photoreactivation studies indicated that pyrimidine dimers were responsible for most of the revertants in RAD+, rad1 and rad6 strains. These experiments show that the RAD6+ locus is intimately concerned with error-prone repair, and suggest that excision repair is substantially error-free.     

Epistatic Interactions of Radiation-Sensitive (rad) Mutants of CAENORHABDITIS ELEGANS

Six double mutants and a quadruple mutant were derived from four UV radiation-hypersensitive single mutants (rad-1, rad-2, rad-3 and rad-7). Sensitivities of the 11 strains to UV, gamma-radiation and methyl methanesulfonate (MMS) were compared. Of the six double mutants, only the rad-1;rad-2 and rad-3;rad-7 doubles were no more hypersensitive than the most sensitive single mutant to UV-radiation. Thus, rad-1 and rad-2 define one epistasis group, whereas rad-3 and rad-7 define another. Consistent with this model was the observation that rad-1 and rad-2, but not rad-3 and rad-7, were hypersensitive to gamma-radiation. In addition, none of the multiple mutants was more hypersensitive to gamma-radiation than the most sensitive single rad mutant. No synergistic interactions of the rad mutations with respect to MMS sensitivities were observed.     

Radiation-Sensitive Mutants of CAENORHABDITIS ELEGANS

Nine rad (for abnormal radiation sensitivity) mutants hypersensitive to ultraviolet light were isolated in the small nematode Caenorhabditis elegans. The mutations are recessive to their wild-type alleles, map to four of the six linkage groups in C. elegans and define nine new games named rad-1 through rad-9. Two of the mutants—rad-1 and rad-2—are very hypersensitive to X rays, and three—rad-2, rad-3 and rad-4—are hypersensitive to methyl methanesulfonate under particular conditions of exposure. The hypersensitivity of these mutants to more than one DNA-damaging agent suggests that they may be abnormal in DNA repair. One mutant—rad-5, a temperature-sensitive sterile mutant—shows an elevated frequency of spontaneous mutation at more than one locus; rad-4, which shows a cold-sensitive embryogenesis, reduces meiotic X-chromosome nondisjunction tenfold and partially suppresses some but not all mutations that increase meiotic X-chromosome nondisjunction; the viability of rad-6 hermaphrodites is half that of rad-6 males at 25°; and newly mature (but not older) rad-8 hermaphrodites produce many inviable embryo progeny. Meiotic recombination frequencies were measured for seven rad mutants and found to be close to normal.     

Interactions Between Light and Gas Vacuoles in Halobacterium salinarium Strain 5: Effect of Ultraviolet Light

The potential light shielding by intracellular gas vacuoles in Halobacterium salinarium strain 5 was examined by looking at the ultraviolet light inactivation curves of both wild-type cells and mutants which are defective in the production of gas vacuoles. Whereas strains defective in gas vacuole production were slightly more sensitive to ultraviolet inactivation, no significant differences in ultraviolet sensitivity were seen, indicating that these subcellular inclusion bodies are not effective as light-shielding organelles. In addition, it was shown that ultraviolet light acts as a plasmid-curing agent in Halobacterium.     

Sedimentation of DNA of Dictyostelium discoideum lysed on alkaline sucrose gradients: role of single-strand breaks in gamma ray lethality of sensitive and resistant strains

The nuclear and mitochondrial DNA of the amoebae of the cellular slime mold Dictyostelium discoideum have been labeled with [methyl-³H]thymidine by allowing them to grow on Escherichia coli 15T^? containing this label in its DNA. Neutral CsCl gradients were used to identify the labeled molecules. Alkaline sucrose sedimentation profiles of cells lysed directly on the gradients revealed two high molecular weight species, one of about 90 S (single-strand mol wt = 1.4 × 10⁸) identified by alkaline CsCl rebanding as nuclear DNA, and another of 43 S (single-strand mol wt = 2.3 × 10⁷), identified as mitochondrial DNA. These alkaline sucrose gradients were used to study the production of single-strand breaks and their rejoining in DNA of a gamma ray-resistant strain (NC-4; 10% survival dose for cell proliferation, D₁₀ = 300 krad) and in two radiation-sensitive daughter mutants (γs-18, D₁₀ = 75 krad; γs-13, D₁₀ = 4 krad). With ⁶⁰Co gamma rays, breaks were produced in nuclear and mitochondrial DNA at an efficiency of one break per 33 eV in all three strains. At doses up to about 100 krad, these single-strand breaks were closed equally well during post-irradiation incubation of NC-4, γs-18 and γs-13, even though their survivals were widely different, indicating no apparent correlation between parental strand rejoining and survival in the sensitive strains. At higher doses, post-irradiation treatment with 1 mg caffeine/ml sensitized NC-4 and retarded strand-rejoining, suggesting that lethality in this resistant strain may be related to strand breaks. It is concluded that single-strand rejoining is a necessary, but not sufficient, condition for radiation survival in this organism. The nature of the apparently unrepaired lesions leading to lethality in the sensitive strains is not known.     

Role of the supX gene in ultraviolet light-induced mutagenesis in Salmonella typhimurium.

Salmonella typhimurium strains with supX mutations are more sensitive than wild type to killing by ultraviolet (UV) irradiation. Studies with strains bearing the leuD21 mutation revealed that inactivation of the supX locus by a nonsense mutation or a deletion results in a complete lack of ability to produce induced Leu+ reversion mutations after UV irradiation. Suppression of the nonsense supX mutation or the presence of an Escherichia coli K-12 F'-borne supX+ allele restored the capacity for induced reversions and increased cell survival after UV irradiation. Introduction of plasmid pKM101 into supX mutant strains also restored their capacity for UV mutagenesis as well as increased survival. The possible nature of the supX gene product and mechanisms by which it may affect expression of the inducible SOS error-prone repair system are considered.     

In vivo nicking and rejoining of nuclear DNA in ultraviolet-irradiated radiation-resistant and sensitive strains of Dictyostelium discoideum

Summary Some aspects of DNA repair in several radiation-resistant and radiation-sensitive strains of Dictyostelium discoideum were investigated by using alkaline sucrose gradients to analyze for the production and resealing of single-strand breaks following irradiation with 254 nm UV. All radiation-resistant strains and all mutants assayed that are sensitive to both UV and ⁶⁰Co gamma rays produced singlestrand breaks in their nuclear DNA after a UV fluence of 15 J/m². Mutants at the radC locus which are sensitive to UV but as resistant as their parental strains to ⁶⁰Co gamma rays produced many fewer single-strand breaks in their DNA after irradiation with UV. Thus, the radC mutations alter a repair pathway specific for UV-induced DNA damage and presumably affect the activity of a UV-damage-specific endonuclease involved in excision repair. All radiation-resistant strains and all of our mutants sensitive to gamma rays rejoined much of their DNA during a three-hour post-UV-irradiation incubation, suggesting that these strains have at least a partially intact excision repair system.Abbreviations used UV ultraviolet light - PBS phosphate buffered saline - cpm counts per minute     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

UV-induced replicating instability in Schizosaccharomyces pombe

A pigmented adenine-requiring strain of S. pombe has been used to study some aspects of the UV-induced replicating instabilities in this yeast. The effect of the repair capacity of a cell on the frequency of such instabilities has been investigated by using three different radiation-sensitive mutants and by studying the influence of caffeine on the frequency of secondary mosaics. Data showed that UV-induced replicating instabilities are not influenced by changes in the repair capacity of the treated cell. Since two of the sensitive mutants used are known to have high spontaneous mutation rates and the third one shows reduced UV-induced mutability, the induction of replicating instabilities differs in this respect both from the induction of spontaneous and UV-induced mutations.     

Comparison of spontaneous, UV-induced, and nitrosoguanidine-induced mutability to drug resistance in myxobacteria.

The UV survival curves of different strains of myxobacteria exhibited shoulders; in the case of Polyangium luteum, an unusual double shoulder appeared. Repair inhibitors like acriflavine, caffeine, and coumarin reduced the survival of UV-irradiated cells if the drugs were incorporated in the post-irradiation plating medium. The shoulders were reduced, but the final inactivation slopes were not affected by the repair inhibitors. Those strains that were resistant to UV were also more resistant to being killed by nitrosoguanidine. A variety of drug-resistant mutants occurred. The spontaneous mutation frequencies to drug resistance varied with the drug and the strain used. Drug-resistant mutants were inducible by UV irradiation and nitrosoguanidine. The UV mutability of Myxococcus xanthus was high compared to Cystobacter sp. However, the nitrosoguanidine mutability of M. xanthus was low compared to the other strains.     

Recombination-deficient mutant of Streptococcus faecalis.

An ultraviolet radiation-sensitive derivative of Streptococcus faecalis strain JH2-2 was isolated and found to be deficient in recombination, using a plasmid-plasmid recombination system. The strain was sensitive to chemical agents which interact with deoxyribonucleic acid and also underwent deoxyribonucleic acid degradation after ultraviolet irradiation. Thus, the mutant has properties similar to those of recA strains of Escherichia coli.     

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.