DNA Repair Dependence of Somatic Mutagenesis of Transposon-Caused WHITE Alleles in DROSOPHILA MELANOGASTER after Treatment with Alkylating Agents

DNA repair-defective alleles of the mei-9, mei-41, mus-104 and mus-101 loci of Drosophila melanogaster were introduced into stocks bearing the UZ and SZ marker sets. Males with the UZ marker set, z1 (zeste allele) and w+(TE) (genetically unstable white allele presumably caused by a transposable element), or the SZ marker set, z1 and w+R (semistable white allele caused by partial duplication of the w+ locus plus transposon insert), were exposed to EMS at the first instar. After emergence, adult males bearing red spots on lemon-yellow eyes were scored as flies with somatic reversions of w+(TE) or w+R. The relative mutabilities (relative values of reversion frequency at an equal EMS dose) of either w+(TE) or w+R in a repair-proficient strain and in mei-9, mei-41, mus-104 and mus-101 strains were 1: approximately 1.2:0.3:0.3:0.7, despite the fact that w+(TE) reverted two to three times as frequently as w+R under both the repair-proficient and repair-deficient genetic conditions. Similarly, after treatment with MMS, MNNG and ENNG, w+(TE) was somatically more mutable in the mei-9 strain and less mutable in the mei-41 and mus-104 strains than in the repair-proficient strain. From these results, we propose that mutagenic lesions produced in DNA by treatment with these chemicals are converted to mutant DNA sequences via the error-prone repair mechanisms dependent on the products of the genes mei-41+ (mei-41 and mus-104 being alleles of the same locus) and mus-101+, whereas they are eliminated by mei-9+-dependent excision repair. In contrast to the approximately linear responses of induced reversions of w+(TE) with ENNG in the repair-proficient, mei-9, and mei-41 strains, seemingly there were dosage insensitive ranges for induced reversion with MNNG in the repair-proficient and mei-41 strains, but not for reversion in the mei-9 strain; w+(TE) in the mus-104 strain was virtually nonmutable with MNNG and ENNG. These results suggest that O6-methylguanine (O6MeG) produced in DNA with MNNG, but not O6-ethylguanine produced with ENNG, is almost completely repaired in a low dose range by constitutive activity of DNA O6MeG transmethylase. From the distribution of clone sizes of spontaneous revertant spots and other data, we propose that both w+(TE) and w+R have a similar tendency to spontaneously revert more frequently at early rather than at later developmental stages probably reflecting a common property of their inserted transposons.     

The white-ivory somatic mutation test in Drosophila. The effects of larval age, increasing the number of copies of wi and the response to some reference mutagens.

The white-ivory somatic mutation test is based on reversion of the X-linked, recessive eye-colour mutation white-ivory to wild-type. Somatic reversion of white-ivory leads to clones of red facets in the white-ivory eyes of eclosing adult flies. The alkylating agents MMS, EMS and ENU and the mutagen DEB all induce high levels of white-ivory reversion. The response of different larval instars has been investigated and dose-response data for MMS and EMS is reported. Strains with additional copies of the white-ivory mutation are more sensitive to reversion but the increase in reversion is not as high as might be expected given the number of white-ivory mutations present. Females, having twice the number of white-ivory copies, tend to show higher levels of reversion than males of the same strain.     

His+ reversions caused in Salmonella typhimurium by different types of ionizing radiation

The yield of his+ reversions in the Ames Salmonella tester strain TA2638 has been determined for 60Co gamma rays, 140 kV X rays, 5.4 keV characteristic X rays, 2.2 MeV protons, 3.1 MeV alpha particles, and 18 MeV/U Fe ions. Inactivation studies were performed with the same radiations. For both mutation and inactivation, the maximum effectiveness per unit absorbed dose was obtained for the characteristic X rays, which have a dose averaged linear energy transfer (LET) of roughly 10 keV/micron. The ratio of the effectiveness of this radiation to gamma rays was 2 for inactivation and about 1.4 for the his+ reversion. For both end points the effectiveness decreases substantially at high LET, i.e., for the alpha particles and the Fe ions. The composition of the bottom and the top agar was the one recommended by Maron and Ames [Mutat. Res. 113, 173-215 (1983)] for application in chemical mutagenicity tests. The experiments with the less penetrating radiations differed from the usual protocol by utilization of a technique of plating the bacteria on the surface of the top agar. As in an earlier study [Roos et al., Radiat. Res. 104, 102-108 (1985)] greatly enhanced yields of mutations, relative to the spontaneous reversion rate, were obtained in these experiments by performing the irradiations 6 h after plating, which differs from the conventional procedure to irradiate the bacteria shortly after plating.     

Unstable ascospore color mutants of Ascobolus immersus

Summary New unstable mutants of Ascobolus immersus involving the color or size of ascospores were sought among spontaneous mutants. Among the 34 unstable mutants isolated, 31 had white spores, 2 had pink spores and 1 had a large sized spores. The unstable mutants involve 11 loci whose mutation leads to white spores and 2 loci whose mutations give pink spores, among the 19 loci known to be implicated in this character; 1 locus is defined by only one large spore mutant. All these genes are localized on at least 7 different chromosomes. Unstable mutants of the same locus may correspond to several different sites, but the number of these sites is very limited.The frequency of unstable mutations was estimated: among 36 mutants belonging to 8 different genes, 20 were stable and 16 were unstable. This high frequency of unstable mutants is undoubtedly underestimated. The moment of reversion of 23 of these new mutants was also sought: 15 of them revert as does mutant B, previously studied, in the very young mycelium, at high temperature and with a reversion frequency of 0.004 to 0.34, according to the mutant; 5 of them revert as mutant 301, also previously studied, during the development of the fruit-body and with a frequency of 0.009 to 0.035; two of these mutants revert very early in the ascospore as soon as the first mitoses or in the very young mycelium at 22° C, with a very high reversion frequency that may reach 1.0; finally, the last mutant studied reverts in the fruit-body with a frequency reaching 0.40, but with modalities different from mutant 301. The mutants of the same locus may revert with different modalities. The same modality may correspond to different sites of the same gene.In unstable double-mutant strains involving two different genes, the reversion of one is independent of the reversion of the other, whether or not the reversion modailities of each mutant are identical.Results indicate the existence of inducers common to several unstable mutants which present the same modalities of reversion.These data support the previously formulated hypothesis of transposable elements.     

Effectiveness of different mutagens in single and combined treatments

An experiment was made on the effectiveness of combined treatments with some chemical (EMS, HA, 5BDUR) and physical (UV, gamma rays) mutagens in inducing visible mutations in hexaploid wheat (Triticum aestivum) variety N.P. 870. Irradiation with gamma rays and chemical treatment with EMS produced a high frequency as well as a wide spectrum of mutations. Treatments with UV, HA and 5BDUR gave a very low frequency of mutations. Combined treatments with any of the latter three mutagens reduced the mutation frequency of gamma rays alone and of EMS alone. The probable reasons for the reduction of the mutation frequency in combined treatments are discussed.     

Reversion of the 8-azaguanine resistant phenotype of variant Chinese hamster cells treated with alkylating agents and 5-brono-2'-deoxyuridine.

From cultures of V79 Chinese hamster cells, 10 independent clones of 8-azaguanine resistant cells were isolated and subcultured. Cells from all ten clones were resistant to 1 mg/ml levels of 8-azaguanine (8-AzG), contained less than 3% of the wild type levels of the enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT), and were unable to grow in HAT medium. The ten clones were classified according to the conditions under which they reverted to the wild type phenotype. Clones in classes I and II reverted spontaneously with frequencies of 40-10(-5) and about 3-10(-5) respectively, and the reversion frequency was independent of the density of cells of all but one of the clones in the culture medium used. Class II clones evinced increased reversion frequencies with ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to a lesser extent with 5-bromo-2'-deoxyuridine (budR), suggesting that these clones contained point mutations in a locus which controls HGPRT activity. The processes of reversion and toxicity appeared to be associated. Class III clones did not revert spontaneously or with BUdR and MNNG, but did revert with EMS. The reversion frequency of class I clones was not increased after treatment with EMS, MNNG or BUdR.     

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.     

Cell-stage dependence of mutagen-induced sister-chromatid exchanges in human lymphocyte cultures

The induction of sister-chromatid exchanges (SCEs) was studied in phytohemagglutinin (PHA)-stimulated human lymphocytes exposed for 1 h to mitomycin C (MMC, 3 X 10(-6) M), ethyl methanesulphonate (EMS, 2 X 10(-2) M), or 4-nitroquinoline-1-oxide (4NQO, 3 X 10(-5) M) at various cell-cycle stages of 72-h cultures. The doses of the chemical were chosen to give about 20 SCEs per cell when treated at Go. The SCE frequency increased almost linearly with MMC or EMS treatments at later times after PHA stimulation, peaking with those at 36 h (at around the first G1/S boundary in the 2 consecutive cell cycles, which was revealed by concomitant experiments), and then decreased with subsequent treatment times. Cell-cycle kinetics and the cell stages at which the cells were treated were measured by autoradiography and sister-chromatid differential staining. The data show that MMC and EMS produce larger numbers of SCEs when treated at stages closer to the beginning of S, and that the most efficient time of treatment is the G1/S boundary in the first cell cycle of the two consecutive cycles before sampling. Pulse treatment with EMS caused about 3 times larger inductions of SCEs when done at late G1/early S(G1/S boundary) in the first cell cycle compared to that at G0/early G1, whereas identical exposure to MMC at the first G1/S boundary produced only 1.5 times larger numbers of SCEs than that at G0/early G1. EMS and MMC both, however, induced 30-40% larger numbers of SCEs when treated at the G1/S boundary in the first cell cycle than when treated at the second cell cycle before sampling. On the contrary, treatment with 4NQO led to the induction of about the same numbers of SCEs even when treated at different cell-cycle stages before the second G1/S boundary. The SCE frequency in 4NQO-treated cells then decreased with subsequent treatment times.     

Modification of the high frequency of reversion of the nicotonic-2 mutant of Coprinus radiatus. II. Polymorphism of strains with low frequencies of reversion (author's transl)]

The nic2 mutation of Coprinus radiatus is unstable at meiosis. Strains derived from the initial mutant can be divided intto two classes: the "neutral genomes" which all revert at meiosis with different frequencies but in an autonomous way; and the "aggressive genomes", which inhibit the reversion of neutral genomes, present a large polymorphism in their own reversion and lose their aggressiveness at meiosis, becoming neutral genomes. The characteristics and the relationships of these two strains are presented in this paper.     

Induced mutations in foxtail millet (Setaria italica Beauv.)

Summary Chlorophyll mutations induced by gamma rays, EMS and DES were studied in foxtail millet (Setaria italica), using two cultures, MU-1 (bristled) and MU-2 (non-bristled). No major differences in the mutagenic response of the two cultures were observed. The treatments included four doses of gamma rays (10Kr, 20Kr, 30Kr, 40Kr) and four durations (6 hrs, 12 hrs, 18 hrs, 24 hrs) each of EMS (0.1%) and DES (0.1%). The combined treatments of gamma rays + EMS and gamma rays + DES were also given.Frequencies of chlorophyll mutations were recorded by three different methods, viz. (a) mutations per cent M₁ plants, (b) mutations per cent M₁ spikes and (c) mutants per cent M₂ plants. No significant differences in the results obtained by these three methods were observed. The frequencies and spectrum of mutations are discussed.Chlorina type were most frequent andviridoalbina least frequent.Striata and virescens were also quite common.Albinos, reported frequently in other crops, were found to be less frequent in foxtail millet during the present study. Number of sectors per spike were also determined from segregation ratios and only one sector per spike was found at all doses. Efficiency and effectiveness of mutagens were also determined and discussed. The results are also discussed with respect to mutagen specificity.     

The multiple-cluster mutation complex in mutagenesis with higher plants

Summary After treatment of dry and pre-soaked seeds of barley with gamma-rays, EMS, NEU and EI, the frequency of multiple mutations (multimutations) was higher with EMS and NEU treatment, while cluster mutations appeared in greater numbers following treatment with gamma rays and NEU. Pre-soaking the seeds led to a reduction in the frequency of total mutations, cluster mutations and multimutations. This has been explained as a result of the application of lower doses and the induction of mutations at a relatively later stage in ontogenetic development in the case of pre-soaked seeds.Some new mutation types in barley have been described and some of the old types have been given names representing the mutation characters more precisely.The compound mutation frequency of different seedling mutation types, when taken separately, was found to be independent of the mutagen employed and the stage of treatment. The size of mutated chimeras in M ₁ plants, as indicated by the segregation ratio of mutants in M ₂, was largest in albina, xantha, chlorina, albina-tigrina, chl-terminalis and eceriferum, and lowest in viridis, viridoalbina etc. This could be expected if the unstable premutations induced by mutagenic treatment are resolved into mutations at different intervals after their initiation, or it can be explained by the induction of dominant mutations, or lethal changes together with visible mutations.     

Efficient Recovery of Enu-Induced Mutations from the Zebrafish Germline

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F(1) progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 X 1 hr in 3 mM ENU and 6 X 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F(2) screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.     

Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Allelic Complementation in the First Gene for Histidine Biosynthesis in SACCHAROMYCES CEREVISIAE. I. Characteristics of Mutants and Genetic Mapping of Alleles

A number of his1 mutants were tested for suppressibility, for reversion by EMS, ICR-170, and nitrous acid, for their allelic complementation response, and for their temperature sensitivity and osmotic remediability. None of 52 mutants tested was suppressible by a known ochre suppressor. This is a very surprising result compared with other studies of suppressibility in yeast and suggests that another function essential to the cell is associated with the his1 gene product, the polarity effect of a nonsense mutation destroying the activity of the his1 enzyme and this second function.Sixty-four his1 alleles were ordered by allelic mapping methods utilizing gamma rays, X-rays, and MMS. The three maps agree well in placement of alleles and have been oriented on chromosome V of yeast with respect to the centromere. The 18 noncomplementing alleles are localized in the distal half of the gene, whereas the complementing alleles are distributed more or less evenly. Mutations which revert to feedback resistance map in the proximal end. Also at this end are mutations having a very high X-ray or MMS induced homoallelic reversion rate. This suggests that a number of missense mutations can occur in this region which result in innocuous amino acid substitutions in the enzyme. One X-ray map unit is estimated to correspond to about 131 base pairs or 43 amino acids, in agreement with results for the cytochrome-c protein obtained by Parker and Sherman (1969).     

Lethals, Steriles and Deficiencies in a Region of the X Chromosome of CAENORHABDITIS ELEGANS

Twenty-one X-linked recessive lethal and sterile mutations balanced by an unlinked X-chromosome duplication have been identified following EMS treatment of the small nematode, Caenorhabditis elegans. The mutations have been assigned by complementation analysis to 14 genes, four of which have more than one mutant allele. Four mutants, all alleles, are temperature-sensitive embryonic lethals. Twelve mutants, in ten genes, are early larval lethals. Two mutants are late larval lethals, and the expression of one of these is influenced by the number of X chromosomes in the genotype. Two mutants are maternal-effect lethals; for both, oocytes made by mutant hermaphrodites are rescuable by wild-type sperm. One of the maternal-effect lethals and two larval lethals are allelic. One mutant makes defective sperm. The lethals and steriles have been mapped by recombination and by complementation testing against 19 deficiencies identified after X-ray treatment. The deficiencies divide the region, about 15% of the X-chromosome linkage map, into at least nine segments. The deficiencies have also been used to check the phenotypes of hemizygous lethal and sterile hermaphrodites.     

Effects of Dose on the Induction of Dominant-Lethal Mutations with Triethylenemelamine in Male Mice

Dose effects of triethylenemelamine (TEM) in the induction of dominant-lethal mutations were studied at the early spermatozoon, midspermatid and spermatocyte stages. The pattern of effects on spermatocytes, unlike midspermatids and early spermatozoa, indicated possible cytotoxic damage, so for the determination of TEM dose-response curves in the induction of genetic damage only the data for midspermatids and early spermatozoa were used. The TEM dose-effect curves for those two stages differ markedly from ethyl methanesulfonate (EMS) dose-effect curves. Beginning with the lowest doses at which significant effects are observed, there is a considerably more rapid increase in dominant-lethal effects with dose of EMS than TEM. Another marked difference between the two compounds is in the ratio of the genetically effective dose (as measured by dominant-lethal mutations) to the lethal dose. The ratio is 1:100 for TEM and only 1:3.5 for EMS; thus, TEM is mutagenic far below its toxic level. Obviously, these results have important implications not only for our understanding of the nature of chemical induction and recovery of chromosomal aberrations but also for the practical problems of evaluating the mutagenic effects of chemicals.     

Population Genetics of LEBISTES (POECILIA) RETICULATUS Peters (Poeciliidae; Pisces). I. Effects of Radiation-Induced Mutations on the Segregation Ratio in Postirradiation F2

Wild-type germ cells of the viviparous fish, Lebistes (Poecilia) reticulatus, were irradiated in the stages of oogonia, spermatogonia or spermatozoa with 1000 R of X rays at a dose-rate of 200-220 R/min. The grown-up fish were mated after irradiation or sham treatment to unirradiated mutant fish homozygous for three recessive gene loci, causing albinism, lack of yellow and reddish pigmentation ("blue" constitution), and diminution of the size of melanophores ("blond" constitution). As compared to the control series, the phenotypic segregation ratios in the adult F(2) generation after irradiation was changed significantly in favour of wild-type and blue fish, whereas the proportion of albinotic, blond and "white" (= blond + blue) guppies was lowered. This unidirectional effect in postirradiation F(2) was greatest after exposure of spermatozoa with 2 x 500 R (24 hr apart), followed by single doses of 1000 R to spermatogonia and oogonia. These results were explained in terms of a synergistic interaction of recessive radiation-induced mutations in the heterozygous state with those genotypes of the guppy which affect the formation of melanophoric patterns and the synthesis of melanins.     

Mutations resistant to bromodeoxyuridine in mouse lymphoma cells selected by repeated exposure to EMS characteristics of phenotypic instability and reversion to HAT resistance by 5-azacytidine

Mutations induced by repeated EMS treatments were investigated by using mouse L5178Y cells. The frequency of TG^r mutations increased linearly with the number of EMS treatments whereas the yield of BrdUrd^r mutations showed a curvilinear dose-response curve. The BrdUrd^r frequency was roughly proportional to the square of the TG^r frequency and the results were compatible with the hypothesis that BrdUrd^r cells were induced by two mutational events within a cell. Most of the BrdUrd^r colonies isolated after 6 EMS treatments, however, were unstable. When BrdUrd^r colonies that had arisen in BrdUrd medium after 2 weeks' incubation were isolated in normal medium, the descendant cells showed a nearly normal level of thymidine incorporation and low plating efficiencies of about 1% in BrdUrd medium. In contrast, after isolation of the same colonies in BrdUrd medium, a low level of thymidine incorporation and high plating efficiencies in BrdUrd medium were observed in the descendant cells.

Reverse selection from BrdUrd^r to HAT^r was accomplished with frequencies of 10⁻⁶−10⁻³ for the descendants grown in BrdUrd medium, and AzaCyd treatment drastically increased the reversion frequency to nearly 10⁻¹. Further re-revertants from HAT^r to BrdUrd^r were also found with frequencies of 10⁻³−10⁻² without treatment. These results indicate that the initial BrdUrd^r cells did not result from inactivation of the thymidine-kinase gene but that the mode of gene expression was altered in some way.     

Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.