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.     

Effectiveness and efficiency of ethyl methanesulphonate and ethylene oxide for the induction of mutations in rice

A comparison of the mutagenic efficiency and effectiveness of ethyl methanesulphonate (EMS) and ethylene oxide (EO) on two different genotypes of rice showed that the mutagenic efficiency sharply decreased with increase in the concentration of EO whereas the efficiency of EMS increased with an increase in the concentration. The effectiveness was inversely proportional to dose of EO whereas it varied little with an increased dose of EMS.     

The detection of mutation in human diploid fibroblasts after mutagen treatment using non-selective cloning and enzyme electrophoresis

In an attempt to study mutagenesis in human diploid fibroblasts, clones derived from mass cultures treated with mutagen have been examined by starch-gel electrophoresis for 43 different enzyme loci. A technique of mutagen treatment was devised which facilitated the cloning and which enabled the cells to be exposed to very high doses of EMS and MNNG. Two alterations in phenotype, presumably the result of mutation, were observed, one involving peptidase D (PEPD) and the other phosphoglucomutase (PGM1).     

A yeast strain for simultaneous detection of induced mitotic crossing over,mitotic gene conversion and reverse mutation

A diploid yeast strain is described which can be used to study induction of mitotic crossing over, mitotic gene conversion and reverse mutation.Mitotic crossing over can be detected visually as pink and red twin sectored colonies which are due to the formation of homozygous cells of the genotype ade240/ade240 (deep red) and ade-2-119/ade2-119 (pink) from the originally heteroallelic condition ade2-40/ade2-119 which forms white colonies.Mitotic gene conversion is monitored by the appearance of tryptophan non-requiring colonies on selective media. The alleles involved are tryp5-12 and trp5-27 derived from the widely used strain D4.Mutation induction can be followed by the appearance of isoleucine non-requiring colonies on selective media. D7 is homoallelic ilv1-92/ilv1-92. The isoleucine requirement caused by ilv1-92 can be alleviated by true reverse mutation and allele non-specific suppressor mutation.The effects of ethyl methanesulfonate (EMS), nitrous acid, ultraviolet light and hycanthone methanesulfonate were studied with D7 stationary phase cells. Mitotic crossing over as monitored by red/pink twin sectored colonies was almost equally frequent among normal and convertant cells. This showed again that mitotic recombination is not due to the presence fo a few cells committed to meiosis in an otherwise mitotic cell population.The dose-response curves for induction of mitotic gene conversion and reversion of the isoleucine requirement were exponential. In contrast to this, the dose-response curve for induction of twin sectored red and pink colonies reached a plateau at doses giving about 30% cell killing. This could partly be due to lethal segregation in the progeny of treated cells.None of the agents tested would induce only one type of mitotic recombination, gene conversion or crossing over. There was, however, some mutagen specificity in the induction of isoleucine prototrophs.     

EMS-induced reversion studies in the white locus of Drosophila melanogaster

5 white-locus mutants of Drosophila melanogaster, representing 5 different sub-sites, were treated with EMS and tested for reversion to wild-type. 4 of them were genuine mutants and one was not. Moreover, the ability of the 4 mutants to revert to wild-type differed from one another which therefore reflects a qualitatively distinct alteration in the genetic material delimited by each mutant.     

Effects of sodium acetate and sodium chloride on EMS-induced chlorophyll mutations in barley

Sodium acetate solutions to which sodium chloride was added, and acetate or chloride alone have been used as pre-, simultaneous, and post-treatment of dry and pre-soaked seeds of barley to study their effect on the types and frequencies of ethyl methanesulphonate (EMS)-induced chlorophyll mutations in spring barley, variety Elsa, and winter barley, varieties $\text{436}\text{35}$ and Ager. Application of acetate/chloride on dry seeds before or simultaneously with EMS both resulted in the frequency of chimeral plants with chlorophyll-deficient sectors in M₁ and chlorophyll mutants in M₂ approximately being halved as compared with the controls (EMS treatment alone).An opposite effect was observed after simultaneous treatment with acetate/ chloride and EMS (pH 4.5 and pH 7.0) and application of acetate/chloride after EMS treatment of pre-soaked seeds. In this case the mutagen sensitivity, i.e. the frequency of chimeral plants with induced chlorophyll-deficient sectors in M₁ and of chlorophyll mutants in M₂, was approximately doubled as compared with the control.Separate application of both acetate or chloride as a simultaneous treatment with EMS resulted also in an increase in the chlorophyll mutation frequency as compared with EMS treatment alone.Based on these results some aspects of the acetate/chloride effect are briefly discussed.     

Methyl methanesulphonate mutagenesis in L5178Y mouse lymphoma cells.

The alkylating agent MMS was toxic to mouse lymphoma L5178Y cells and decreased their growth rate. A dose-dependent induction of thioguanine- and thymidine- but not ouabain-resistant variants was observed. The prolonged period for expression of thioguanine-resistant variants observed with other mutagens was also found in these studies. A comparison of MMS and EMS showed that MMS on a molar basis was approximately 10 times more toxic than EMS. With mutation, however, when evaluated at equal levels of cell killing MMS and EMS induced the same number of thymidine-resistant variants. For thioguanine-resistant variants MMS was approximately 10-fold less efficient than EMS, while for ouabain-resistance MMS, unlike EMBS, produced no variants at all. The ouabain results were further compared with positive results obtained using a modified Luria--Delbrück fluctuation test.     

Heritable translocation test and dominant-lethal assay in mice with methyl methanesulfonate.

A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F1 males varied between 2 and 99%. Transmission of partial sterility and translocations was confirmed in the F2 generation. There were no partially sterile or sterile males among the 245 controls.     

Alkylation of cytosine and 5-hydroxymethyl-cytosine by methyl methanesulphonate and N-methyl-N-nitrosourea: its relevance to mutagenesis.

The reaction of cytosine and 5-hydroxymethyl-cytosine (OHMeCyt) with a variety of monofunctional alkylating agents has been investigated to evaluate further the possible role of cytosine alkylation in mutagenesis and the possibility that the immunity of T-even phages to mutation by methyl methanesulphonate (MMS) was due to the unreactivity of OHMeCyt towards this agent. Both cytosine and OHMeCyt reacted equally well with the methylating agents MMS and N-methyl-N-nitrosourea (MNU) affording 6% and less than 1% respectively of the 3-substituted derivative. No product was isolated following subjection of the bases to reaction with ethyl methane-sulphonate (EMS), N-ethyl-N-nitrosourea (ENU) or iso-propyl methane-sulphonate (iPMS).     

Inactivation and mutation of coliphage T4 by aliphatic nitrosamides and methanesulphonates: in vitro recovery of infectivity of T4 inactivated by isopropyl methanesulphonate.

The inactivation and mutation (to r phenotype) of extracellular coliphage T4 wild-type by the monofunctional alkylating agents N-methyl- and N-ethyl-N-nitrosourea and isopropyl methanesulphonate were investigated. The rate and extent of change in phage infectivity observed during the post-treatment period were found to correlate with what is known of the mechanisms by which these agents react in vitro. Loss of phage infectivity was found to occur during the period following treatment with these agents, but that resulting from treatment with isopropyl methanesulphonate was preceded, in the first 24 to 48 h, by a recovery of infectivity. This suggested that changes in phage infectivity occurring after treatment with monofunctional alkylating agents are resultant of various processes which diversely promote loss and recovery of infectivity. The mutagenicity of N-methyl-N-nitrosourea was similar to that of its ethyl homologue at a level of phage survival of 4 x 10-3, but less than that of isopropyl methanesulphonate. At a level of survival of 3 x 10-2 ethyl methanesulphonate was a mutagenic as its isopropyl homologue, but methyl methanesulphonate was only slightly if at all mutagenic. These results could not be correlated with the compounds' reaction mechanisms. The efficiency of isopropyl methanesulphonate (compared with its toxicity to phage) was found to decrease as the severity of the dose was increased.     

Factors affecting the quantitation of dose-response curves for mutation induction in V79 Chinese hamster cells after exposure to chemical and physical mutagens.

Using four common mutagens, ethyl methanesulphonate (EMS), methyl methanesulphonate (mms), uv, and X-irradiation, the relationship between dose of mutagen, cellular lethality and frequency of 8-azaguanine resistant colonies in V79 Chinese hamster cells has been examined. Several factors affecting the recovery of mutants including inter and intra-clone metabolic co-operation have been quantitated and their influence on survival response curves examined. Induced mutant frequencies were assayed by two methods in situ, and after replating. After exposure to X-rays, MMS and UV a significantly higher frequency of mutants was observed in replated experiments as compared with the in situ situation, at all survival levels assayed. With EMS, an increment on replating was observed only at high survival levels. The replating data suggest that two types of azgr colonies are produced, i.e. those which contain only azgr cells and those which, due to damage segregation, contain a mixture of azgr and azg8 cells. These mixed colonies appear to be lost by metabolic co-operation when mutation frequencies are assayed in silu. The proportion of mixed to homogeneous colonies differs with different mutagens. Taking into account such factors, EMS and UV irradiation were similarly mutagenic at a given survival level, but at equitoxic doses, fewer mutants were recovered after exposure of V79 cells to MMS and X-rays.     

A comparison of mutation induction by 14 MeV fast neutrons and 137 Cs -rays in meiotic spermatocytes in the silkworm

Barley seeds were treated with methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), stored at 15% water content and washed for 16–24 h. These treatments resulted in an increase of toxic and genetic effects. In teh DNA of embryos of such stored MMS- and EMS-treated seeds, a strong enhancement of the amount of single-strand breaks and/or alkali-labile sites took place. In contrast, the amount of alkylated sites, particularly of 7-methylguanine, was somewhat lower. It can be that the depurination and/or backbone breakage, which proceeds during the storage period, is responsible for the enhancement of toxic and genetic effects, whereas the influence of the alkylation of DNA during the storage period by the unreacted residual mutagen is negligible.     

Testing of some permitted food colours for the induction of gene conversion in diploid yeast.

12 permitted food colours in use were screened for geno-toxicity. Mitotic gene conversion in Saccharomyces cerevisiae was used as the end-point. Each food colour was tested in stationary-phase as well as log-phase cells but without microsomal activation. These food colours did not cause any increase in mitotic gene conversion in diploid yeast BZ 34.     

Mutagenic effect of BUdR in diploid human fibroblasts

It has only recently been possible to demonstrate the expected mutagenic effect of 5-bromodeoxyuridine (BUdR) in heteroploid hamster cells in culture. We have now extended this observation to diploid human fibroblasts utilizing techniques adapted from the work of Albertini and DeMars on X-ray mutagenesis at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in these cells. In four separate experiments, fibroblasts from a female donor were exposed to 500 micrograms/ml ethylmethane sulfonate (EMS) or 3 micrograms/ml BUdR yielding survivals of 9% and 5%, respectively. After a 6-day expression period, survivors were plated in selection medium containing 0.3 micrograms/ml 8-azaguanine (8-AG). After 3-5 weeks, azaguanine-resistant colonies were isolated for characterization or stained for counting. The average spontaneous mutation rate/cell/generation was 0.6.10(-6). The average induced mutation rates for EMS and BUdR were 7.8.10(-6) and 6.3.10(-6)/cell/generation, respectively. Similar results were obtained in two experiments with an additional fibroblast line. Mutant colonies isolated following BUdR treatment demonstrated from 1.4 to 61.5% of the HGPRT activity of the parental line and showed at least 8% Barr bodies, excluding the possibility of contamination by Lesch-Nyhan cells. This demonstration of a BUdR effect comparable to that of an alkylating agent or X-irradiation opens the study of mutation due to base-analog substitution in diploid human cells.     

Mutagenesis by nitrosoguanidine, ethyl methanesulfonate, and mutator gene mutH in continuous cultures of Escherichia coli.

Induction of T5-R mutations by alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS) was examined in glucose limited chemostat cultures of non-mutator and mutator (mutH) bacteria. In agreement with the proposal that NTG mutagenizes DNA at the replication fork, this mutagen (6.8 X 10-minus 6 M) showed replication-dependent mutagenesis in continuous culture. EMS (5-10-minus M)) induced mutagenesis could not be correlated with growth rate, which probably means that induction of mutagenic lesions (promutations) by this mutagen does not involve replicating genes. A large synergic response was found for the mutH gene in combination with NTG, supporting the hypothesis that the mutH gene product acts during DNA replication.     

The nature of reverse mutations in Drosophila melanogaster

A selection system for the screening of reversions has been constructed and used to test reversions of lethals located in the proximal region of the X chromosome of Drosophila and of Kpn mutations.Spontaneous and induced reversions have been screened, X-rays and ethyl methanesulphonate (EMS) being the mutagens used in the induction experiments.No genuine back-mutation was found in 6·10⁵ gametes scored. Sterile reversions of all four lethals tested were obtained. Their frequency suggested that at least in three of the lethals the sterile reversions represented “escapers” of the lethal effect rather than true revertants.Three fertile reversions of l^x4 were found and analyzed. All three were autosomal suppressors, located on the second chromosome, allelic to each other, dominant in males and recessive in females.One fertile reversion of l^3DES was found to be an X-linked suppressor. It is suggested that this suppressor is a Y-suppressed lethal, showing a V-type position effect, resulting from an aberration included in the proximal heterochromatin of the X chromosome.Reversions of Kpn were obtained at a similar rate to that found in previous reports²².The absence of true back-mutants in our experiments, in contrast to findings in previous reports, is discussed. From the existing literature on spontaneous and induced back-mutations in Drosophila melanogaster it appears that for several mutations the rates of forward and back-mutation are of the same order of magnitude. It is suggested that reported cases of back-mutations represent mainly inter- and intrachromosomal recombination in duplicated regions rather than mutational events and that the frequency of true back-mutation in Drosophila is usually of an order of magnitude, similar to that known for microorganisms and fungi.     

Repair processes and the response of dividing and non-dividing cells to methyl methanesulphonate and dimethyl sulphate.

A method for producing a viable non-dividing population of Chinese hamster V79 cells in suspension is described and the characteristics of the population outlined. The stationary population is more sensitive to methylating agents than a similar but exponentially growing population, the increased sensitivity arising from the loss of the shoulder from the survival curve. The extent of reaction of the agent with cellular macromolecules is similar in both cases. The repair capabilities of the two populations was examined. Non-semiconservative DNA repair synthesis occurs whether the cells are in a growth or no-growth condition when insulted. Repair of single-strand breaks, which arise following methylation, also proceeds up to the size of the replicon. The relationship of this stationary population to other no-growth conditions and its utility as a model for carcinogenesis studies is discussed.