Synergism and antagonism between N-methyl-N'-nitro-N-nitroso-guanidine and 9-aminoacridine in mutation induction in Escherichia coli K12.

When bacteria were treated simultaneously with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and low concentrations of 9-aminoacridine (9AA), the yield of frameshift mutations was much greater than that expected on the basis of independent action of both mutagens. In combination with a high concentration of 9AA, however, MNNG had an antagonistic effect upon the induction of frameshift mutations. There was no synergistic interaction between the two mutagens in bacteria in which the adaptive response to methylating agents had been induced. 9AA not only induced frameshift mutations, but also caused a small increase in reversions of a nonsense (ochre) mutation and, in combination with low MNNG concentrations, it had a small synergistic effect.

Strong antimutagenic effect of caffeine on 9-aminoacridine-induced frameshift mutagenesis in Escherichia coli K12.

Caffeine, present at a concentration of 3 mg/ml during treatment with 100 microM 9-aminoacridine (9AA) of wild-type Escherichia coli K12, caused a decrease in the yield of frameshift reversions of more than three orders of magnitude. Antimutagenesis of caffeine was shown to be due to partial inhibition of induction by 9AA of frameshift replication errors, resulting in an increased efficiency of mismatch repair of the pre-mutations produced under these conditions.

Induction of frameshift mutations by caffeine in Escherichia coli K12.

Caffeine induced reversions of five different frameshift mutations in Escherichia coli K12. Only those mutations which were very sensitive to reversion-induction by 9-amino-acridine (9AA) were also sensitive to caffeine. Caffeine mutagenesis was not due to inhibition of cellular repair functions, but to the production of replication errors, which were corrected very efficiently by mismatch repair. From the results reported here we conclude that the targets for caffeine mutagenesis are long runs of GC base pairs, and that this mutagen induces -1 frameshifts.

On the glucose effect in acridine-induced frameshift mutagenesis in Escherichia coli.

Log-phase cells of E. coli growing in defined minimal media were washed, exposed to acridines in the same minimal salts solution, and plated to select for Nad+ revertants. At low mutagen concentration, treatment in the presence of the carbon source to which the cells were adapted resulted in a decrease in revertant yield of several orders of magnitude compared with the yield in the absence of a carbon source. At high mutagen concentration, however, a carbon source present during treatment caused a 2- to 150-fold increase in revertant yield (depending on the mutagen, the carbon source, and on the genetic background of the strain). In a strain lacking adenylate cyclase, acridine mutagenesis was not abolished under the experimental conditions used in this study, and the addition of cAMP during mutagenic treatment had no effect. In mismatch repair-deficient strains, the presence of glucose during treatment with low mutagen concentration did not cause a decrease in revertant yield as drastic as in the wild type. From the results reported here, we conclude that the glucose effect in acridine mutagenesis is due to an enhancement of mismatch repair.

Evidence for frameshift mutations in the hisH gene of Escherichia coli causing synthesis of a partially active glutamine amidotransferase.

Among eight strains carrying acridine-induced mutations in hisH, five which mapped at four different sites in the promoter-distal region of the gene showed His+ phenotypes on media containing a purine. By complementation analysis, hisH enzyme was shown to be required for growth on purines. Purine-sensitive His+ revertants of strains able to grow on purines carried second-site mutations which in one case could be shown to map in hisG. Strains able to grow on purines were able to grow on 2-thiazolyl-DL-alanine, too. We conclude that frameshift mutations in the promoter-distal part of the hisH gene of E. coli do not completely abolish the activity of the gene product.

Genetic analysis of clear-plaque mutations induced in bacteriophage lambda by 9-aminoacridine.

Clear-plaque mutations were induced in the cI and cII genes of lambda by treating lysogenic cells with 9-aminoacridine (9AA). Mapping of the mutations revealed that there were two hot spots for 9AA mutagenesis in cI, and one strong hot spot in cII. The hot spots in cI mapped close to 1 of the 3 runs of 4 G/C base-pairs and near the only run of 5 G/Cs, respectively, in this gene. Of 36 cI mutations tested, at most one mapped near a run of 6 A/T base-pairs. By analogy, the sequence responsible for the strong hot spot in cII may be the run of 6 G/Cs in this gene.

Methodologies for the determination of various genetic effects in permeable strains of E. coli K-12 differing in DNA repair capacity. Quantification of DNA adduct formation, experiments with organ homogenates and hepatocytes, and animal-mediated assays.

Derivatives of E. coli K-12 strain 343/113 differing in DNA repair capacity, in permeability to large molecules, and in some metabolizing activities (nitroreductase, glutathione), were constructed for the quantitative determination of the induction of various genetic effects, such as forward and back mutations, lysogenic induction of prophage lambda, and repairable DNA damage. These E. coli strains can be used in assay procedures which allow variation and control over several experimental conditions, such as oxygen tension, time, pH, temperature of incubation and growth phase of the indicator cells. Methods are described for the simultaneous determination of genetic effects and of DNA-adduct formation during mutagen treatment, i.e. by using radio-labeled compounds or by means of an enzyme-linked immunosorbent assay (ELISA). Mammalian biotransformation of xenobiotics can be investigated by including various fractions of mammalian organs in the system. Examples of the relative effectiveness of the activating potential of S9, S100 and isolated hepatocytes for dialkylnitrosamines and other carcinogens are presented. Host-mediated assays, finally, are described which, in addition to gene mutations, can also be used for the determination of repairable DNA damage in bacteria present in different organs, including the liver, spleen, lungs, kidneys, pancreas, and the blood stream of chemically treated mice. It is concluded that quantitative tests in vitro for assessment of induced mutagenic spectrum and genotoxic potency, combined with the host-mediated assay as a monitor, in vivo, of genotoxic factors present in various organs of animals, may become useful in the assessment of genotoxic (and possibly tumor-initiating) properties of chemicals for which long-term in-vivo mutagenicity and/or carcinogenicity data are not yet available.

Mutation induction by thymine deprivation in Escherichia coli B/R. II. Mutation in the repressed and derepressed tryptophan operon.

True Trp+ reversions are induced by thymine deprivation in cells with repressed trp operons as efficiently as in derepressed cells. At least part of the mutations are fixed during thymine starvation, i.e. in the absence of net DNA synthesis. The hypothesis is put forward that thymineless mutagenesis is due to repair-replication under limited concentrations of 5'-dTTP, performed by an inducible error-prone "DNA-polymerizing activity" on single-strand gaps.

Mutation induction by thymidine deprivation in Escherichia coli B/r. I. Influence of caffeine.

The addition of caffeine to the plating medium after thymine deprivation of E. coli WP2 uvr+ thyA or WP2 uvrA thyA had no influence on survival. Caffeine, however, reduced the frequency of mutants. The hypothesis is presented that the reduced mutagenesis is due to the sensitivity to caffeine of an inducible error-prone repair mechanism operating during thymine deprivation and after the re-addition of thymine.