Factors affecting the growth of Chinese hamster cells in halt selection media

Factors affecting the efficiency of selection of “reverants” of salvage pathway mutants in media containing amethopterin have been examined. Our V79 Chines hamster cell line was found to require a significantly higher level of thymidine for optimal growth in such media than has been reported for other cell lines. Hypoxanthine (but not glycine) was also required for reversal of amethopterin toxicity, but levels did not differ significantly from those reported elsewhere. Growth in HAT was also dependent on plating density and serum batch. Our modification (VHAT) was compared with published HAT recipies in back selection reconstruction experiments. A sharp fall in EOR (efficiency of recovery) of wild type cells from mixtures with mutants at plating densities greater than 3500 cells/cm² (10⁵ cells/6 cm dish) was observed for VHAT. EOR with other HAT recipes was lower still, and was affected also by the particular mutant used in the mixture.EMS induced “revertants” were isolated from three 8AZ^r mutants by plating in VHAT. All. revertants were however amethopterin resistant, they were also 8AZ resistant and the mobility of residual HGPRT (as measured by polyacrylamide gel electrophoresis) was similar to that of their 8AZ^r parents i.e. dissimilar from that in wild type. The modal chromosome number of V79 wild type cells was 21. No significant deviation from this mode was detected in any of the mutant lines examined. The data indicate that the recovery of colonies in HAT from 8AZ^r mutants does not necessarily indicate that a back mutation in the structural gene for HGPRT has occurred. Thus, the frequency of HAT⁺ colonies cannot be taken as a direct indication of reversion frequencies.     

Effects of chemical and physical mutagens on the frequency of a large genetic duplication in Salmonella typhimurium. II. Stimulation of duplication-loss from merodiploids.

Strains of Salmonella typhimurium which contain a duplication of approximately 30% of the genome may be obtained by a simple selective procedure. These strains are highly unstable, losing the duplication when grown on non-selective medium. In this paper we report that treatment of merodiploid bacteria with mutagenic agents stimulates the rate at which haploid segregants are obtained from merodiploid strains. The mutagens which have been tested for this effect are X-rays, ultraviolet light (UV), ethyl methanesulfonate (EMS), and the azaacridine half-mustard ICR-372.     

Bone marrow and thymus regeneration is a condition for thymoma development

Degeneration and regeneration of bone marrow was measured by nucleated cell number changes and of thymus and spleen by weight changes in adult female inbred SwisS mice given a single sub-lethal dose of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), or N-ethyl-N-nitrosourea (ENUA).Significant cell number decreases of the bone marrow were observed only following ENUA, the only one of the four agents capable of enhancing thymoma development in these mice. ENUA also caused the greatest significant weight decrease of the thymus and the spleen. Return to normal occurred before the 20th day of the experiment.It is suggested that bone marrow and thymus regeneration is an essential step in thymoma development.     

Evidence for an increase in X-ray-induced translocation frequency in oocytes of caffeine-treated Drosophila females

0-8 h old Drosophila females carrying a reversed metacentric X chromosome and a suitably marked Y chromosome were treated or not with 0.2% caffeine and irradiated with 2000 R X-rays. In contrast with the reduction found in translocation frequency following 2000 R irradiation of the male mated with 0.2% caffeine-treated females, the frequency of interchanges in oocytes was significantly higher with caffeine as compared with controls.     

Genetic toxicity of procarbazine in bacteria and yeast.

Procarbazine [N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride] is used to treat Hodgkin's disease. This compound was tested in vitro without and with S10 fraction from mice liver (microsomal assay) using Saccharomyces cerevisiae strain D7, Salmonella typhimurium (strains TA98, TA100, TA1535) and in vivo in Swiss albino mice (host-mediated assay) using D7. Procarbazine, without S10 fraction, is highly toxic and induced mitotic crossover, gene conversion, and reverse mutation in D7. It had a toxic effect on all the Salmonella strains; but did not induce reverse mutations at the histidine loci. Procarbazine, with S10 fraction, was less toxic and did not induce genetic effects in yeast or Salmonella. In the host-mediated assay, no genetic effects were seen.     

Induction of dominant-lethal mutations after administration of ethylenethiourea in combination with nitrite or of N-nitroso-ethylenethiourea in mice

Mutagenic potentials of ethylenethiourea (ETU) in combination with sodium nitrite or of N-nitroso-ETU, a nitrosttion product of ETU in vitro, were investigated in the mouse dominant-lethal test. Simultaneous 5-day p.o. administration of ETU (150 mg/kg) and sodium nitrite (50 mg/kg) caused a significant decrease in the percentage pregnancy and the number of implants in weeks 5 and 6 of testing, although no effects were obtained on these indices when the chemicals were applied separately. However, in the group treated with 30 mg ETU plus 10 mg sodium nitrite per kg no dominant-lethal mutations were induced. 5-day oral administration of 100 mg of N-nitroso-ETU per kg also exhibited similar effects to those observed after treatment with 150 mg ETU plus 50 mg sodium nitrite per kg.     

Mutagenicity testing and risk estimation with mammals

Mammalian test systems are currently used for mutagenicity screening. The necessity and the limitations of standardizing these methods are discussed for the dominant-lethal assay. In addition to the refinement of standard methods, the development of new systems in mammals is emphasized. One promising approach is the detection of presumed somatic mutations. Another new development takes advantage of electrophoretic methods for detecting induced structural alterations of gene products. Mammalian experiments will be essential for the assessment of risks from chemical mutagens. The development of standards for the controlled use of chemical mutagens should be guided by the experience accumulated in radiation genetics. Two methods, the measurement of specific-locus mutation rates in mice and the direct determination fo the phenotypic damage of dominant genes affecting the skeleton of mice, are recommended for the assessment of the hazard of chemical mutagens.     

Analysis of Variety Trials in the Presence of Non-Random Time Effect

The assumption of random year effect in the analysis of variety trials data over locations and years clearly contradicts the practice of conducting the trials over only a few consecutive years. A method of analysis of such trials which assumes that the year-dependent effects are serially correlated is proposed in this paper to cater for the non-randomness in the selection of years of the experiments. A simple autoregressive model was assumed for the serial relationship. A reduction in the variance estimate of the difference between two variety means was obtained using this method.     

Dose and effect of methyl-2-benzimidazolylcarbamate in the "mammalian spot test", an in vivo method for the detection of genetic alterations in somatic cells of mice.

In the spot test, mouse embryos which are heterozygous for four different recessive coat-colour genes are treated in utero by injection of a mutagen into the peritoneal cavity of the mother or by other appropriate routes of administration. If this treatment leads in a pigment precursor cell to an alteration of the wild type allele of one of the genes under study or to its loss, a colour spot in the adult coat may be seen. Peroral application of 100-300 mg methyl-2-benzimidazolylcarbamate (MBC)/kg to the mother during the tenth day postconception led to an increase in the frequency of colour spots in the coats of offspring. The data are consistent with the hypothesis that MBC is a point mutagen.