An Escherichia coli mutant refractory to nitrosoguanidine mutagenesis

Summary A newly-isolated Escherichia coli mutant suffers only about 10% as many mutations as normal strains on exposure to nitrosoguanidine¹. The responsible mutation, inm-1, maps at approximately minute 79 in the current E. coli genetic map. The mutant is normal for overall growth, nitrosoguanidine lethality, spontaneous mutagenesis, ultraviolet light lethality and mutagenesis, ethyl methanesulfonate lethality and mutagenesis, and the adaptive repair induced by alkylating agents. The existence of this mutation proves that nitrosoguanidine mutagenesis is not merely the result of reactions between the chemical and DNA, but requires specific cellular function(s), and underscores the peculiarity of nitrosoguanidine as a mutagen.     

Chromosomal location of a gene (idg) that specifies production of the blue pigment indigoidine inErwinia chrysanthemi

Production of a blue pigment, indigoidine, is a variable trait among wild-type strains ofErwinia chrysanthemi; it is also influenced by the composition of the growth medium. Starting with a nonpigmented wild-type strain (ICPB EC183) ofE. chrysanthemi, we obtained by nitrosoguanidine mutagenesis a pigmented (idg ⁺) mutant strain (AC6055), which simultaneously was Arg^–. Linkage betweenarg andidg was established in two-factor transductional and conjugational crosses. Coinheritance ofidg withhis ⁺,ilv ⁺,leu ⁺,ser ⁺,thr ⁺, orura ⁺ transductants was not observed. Spontaneous Arg⁺ revertants of AC6055 were invariably Idg⁺. The pigments produced by AC6055, Idg⁺ recombinants, and wild-type Idg⁺ strains were identical, judged by absorption spectra (_max = 615 nm) of the dimethylsulfoxide extracts of whole cells. We concluded that nitrosoguanidine caused comutation in thearg andidg loci that are linked on theE. chrysanthemi chromosome.     

Lethality and mutagenicity inHalobacterium mediterranei caused by N-methyl-N′-nitro-N-nitrosoguanidine

Compared withEscherichia coli, Halobacterium mediterranei was highly resistant to the lethal effect of N-methyl-N-nitro-N-nitrosoguanidine (nitrosoguanidine), but it was sensitive to the mutagenic action of this chemical agent. Nitrosoguanidine at 500 g ml^–1 gave a cell survival level between 1% and 10%, and this allowed us to obtain more Josamycin-resistant mutants compared with lower concentrations, which gave higher survival rates but fewer mutants. The efficiency of the mutagenicity obtained with the nitrosoguanidine treatment was examined under a variety of conditions. The optimal conditions for obtaining Josamycinresistant mutants were achieved by exposing, in darkness and without shaking, a suspension of about 10⁸ log-phase cells to 500 g nitrosoguanidine in 1 ml of 50 mM modified saline Tris-maleate buffer at pH 7.5, or in 1 ml of 5 mM modified saline Tris-citrate-maleate for 30 min at 37°C.     

Replicating instabilities in yeast: occurrence in different mutational systems

Summary Following mutagenesis of yeast cells with nitrosoguanidine, primary mosaic colonies exhibiting prototrophic/auxotrophic phenotypes were obtained. Upon replating of these primary mosaics, numerous secondary mosaics were present in the progeny. This study shows that replicating instabilities occur at many different loci within the Schizosaccharomyces pombe genome. In addition, the ade-1 gene of Saccharomyces cerevisiae (causing red pigmentation) was used to show that the phenomenon also occurs in this yeast.NRCC#240/8     

The effect of nitrosoguanidine upon DNA synthesis in vitro

Summary Both the polymerase and the exonuclease activities of DNA polymerase III^* are inactivated by treatment with nitrosoguanidine. The treatment of the DNA template with the mutagen does not affect the template in supporting DNA synthesis. No effect of nitrosoguanidine upon fidelity of replication in vitro was detected.     

The action of nitrosoguanidine and other DNA-inactivating agents on Streptococcus pyogenes K56 strains with normal and reduced dark repair ability

Summary The response pattern for ultraviolet light, nitrogen mustard, and ethyl methane sulphonate of Hcr⁺ and Hcr^- strains ofStreptococcus pyogenes K 56 is similar to that observed for analogous strains ofE. coli, whereas repair-apt streptococcal strains are much more sensitive to nitrosoguanidine and mitomycin C thanE. coli. Theuvr gene(s) appear(s) to be without effect upon survival, prophage induction, and mutation to streptomycin resistance caused by nitrosoguanidine and only of little influence on repair of mitomycin C damage.     

Mutagenesis byN-methyl-N-nitroso-N-nitroguanidine in synchronized cultures ofMycobacterium phlei

Conditions of maximum induction of back mutations byN-methyl-N-nitroso-N′-nitroguanidine (“nitrosoguanidine”) were studied in auxotrophic mutants ofMycobacterium phlei. In asynchronous cultures the effects of pH, buffer molarity and concentration and exposure time to nitrosoguanidine were studied. It was shown that between 6 and 10, pH does not affect the induction of back mutations but that with increasing pH up to 9 the lethal effect of nitrosoguanidine on cells is increased. Protracted treatment with nitrosoguanidine or buffer molarity did not affect the induction of back mutations. It was found with several strains ofMycobacterium phlei that it is most efficient to treat a culture with 0.5 mg or 1 mg nitrosoguanidine/ml for 20 min at pH 6. On the basis of these findings a method of induction of back mutations by nitrosoguanidine was developed for populations with synchronous cell division.     

First identification of an amber nonsense mutation in Schizosaccharomyces pombe: major differences in the efficiency of homologous versus heterologous yeast suppressor tRNA genes

Summary In Saccharomyces cerevisiae ochre and opal, as well as amber mutations are known, whereas in the fission yeast Schizosaccharomyces pombe no amber alleles have been described. We have characterized trp1-566, an amber allele in the trp1 locus of S. pombe. The identification of trp1-566 as an amber allele is based on the following results: (a) The nonsense allele can be converted to an ochre allele by nitrosoguanidine mutagenesis. (b) trp1-566 is suppressed by a bona fide S. pombe amber suppressor tRNA, supSI. The supSI gene was obtained by primer-directed in vitro mutagenesis of a tRNASer from S. pombe. Unexpectedly, an S. cerevisiae amber suppressor tRNASer, supR21, transformed into S. pombe, failed to suppress trp1-566. Northern analysis of S. pombe transformants, containing supRL1 or S. cerevisiae tRNA^Leu or tRNA^Tyr genes reveals that these genes are not transcribed in the fission yeast. As an additional tool for the analysis of nonsense mutations in S. pombe, we obtained by nitrosoguanidine mutagenesis two unlinked amber suppressor alleles, sup13 and sup14, which act on trp1-566.     

A relaxed mutant with an altered ribosomal protein L11

Summary A replica plating method for isolating ts amoebal mutants of Physarum polycephalum has been devised. Temperature-sensitive mutations occur at a frequency after nitrosoguanidine mutagenesis of 10^-3 per survivor, are stable but are not usually expressed in the plasmodia formed from these amoebae in clones. Some of these mutants appear to be cell-cycle stage specific.     

Escherichiacoli mutants deficient in exoribonucleases

Strain S296, isolated by screening 2000 colonies after nitrosoguanidine mutagenesis, yields extracts with less than 1% of wild-type RNase activity against (³H) poly(U). Unlike other $\text{E.}\text{coli}$ strains, S296 grows with a doubling time of about 2 hr., both in nutrient broth and in minimal medium, and at 30°, 37° and 42°. The strain retains 10 to 20% of wild-type exonuclease activity against (³H) rRNA or T4 phage-specific mRNA; but two further mutants, made by screening mutagenized colonies of strain S296, are reduced to 3% of wild-type activity against those substrates as well.     

Reversion from Suppression to Nonsuppression in SUQ5 [psi+] Strains of Yeast: The Classification of Mutations

Reversion from the suppressed to nonsuppressed phenotype in strains of genotype SUQ5 [psi⁺] ade2-1 his5-2 lys1-1 can1-100 ura3-1 has been induced by treatment with ethyl methanesulphonate, nitrosoguanidine or UV (254 nm) light. Spontaneously occurring revertants have also been selected by two different methods. Reversion has been shown to occur through a variety of nuclear mutations and through mutation of [psi⁺] to [psi^-]. Nuclear mutations included back-mutation of SUQ5, antisuppressor mutations that were recessive, semi-dominant or dominant, and dominant or recessive mutations of genes required for the maintenance of the [psi⁺] factor. Complementation tests by which the various kinds of mutations could be distinguished from one another were designed. The spectra of spontaneously occurring and induced mutations have been described.     

The mutagenic effect of nitrosoguanidine onMycobacterium phlei PA

The effect of nitrosoguanidine on the induction of three types of mutagenic changes inMycobacterium phlei PA was studied. The mutagenic changes included: change of prototrophy to auxotrophy, conversion of sensitivity to isoniazide to resistańce and sensitivity to streptomycin to resistance. The induction of auxotrophic mutants was successful especially when using NTG at a concentration of 1000 μg/ml. A total of 100 auxotrophs was obtained out of which only 13 were sufficiently stable to be used in further studies. Amino acid requirements especially the glycine⁻(serine⁻) type characterized more than half of all auxotrophic mutants obtained. A group of mutants requiring purines also included a high number of mutants. A considerable spontaneous reversion frequency was found in both groups of auxotrophs. The kinetics of the induction of INH-resistant mutants by nitrosoguanidine at a concentration of 1000 μg/ml was studied and a high induction of these mutants, particularly at high lethal effect of the mutagen found. The mutability of the STM^r marker was relatively low in the present model microorganism as compared with the two markers mentioned earlier.     

The effects of mutations in the polA and recA genes on mutagenesis by nitrosoguanidine in Salmonellatyphimurium

Results of semi-quantitative plate tests indicated that polA and recA mutants of Salmonella typhimurium strain LT2 trpB1 might be significantly less mutable by nitrosoguanidine (MNNG) than were their repair-proficient parents strains. Quantitative data obtained in treat-and-plate experiments showed that this was not the case, at least for low doses of MNNG, and also that the recA strain was significantly more mutable at low doses than its Rec⁺ parent. On the basis of these results it is suggested that cells of S. typhimurium may possess a recA⁺-dependent repair pathway capable of error-free removal of MNNG-induced pre-mutational lesions from their DNA.     

Clostridium acetobutylicum mutants isolated for resistance to the pyruvate halogen analogs

Pyruvate halogen analogs, 3-fluoropyruvate and 3-bromopyruvate, are toxic towardClostridium acetobutylicum. After mutagenesis with nitrosoguanidine, mutants resistant to these compounds were selected. In a normal batch culture regulated at pH 4.8, mutants quickly initiated the acid-solvent transition, accumulating more acetoin and lactate than the wild type strain. The maximum rate of specific glucose uptake was higher in the mutants than in the wild type: 1.76 h^–1 and 0.9 h^–1 respectively. When the pH was uncontrolled, mutants converted glucose into solvents, essentially butanol, while the wild-type strain ceased its fermentation at the acidogenic stage. Enzymatic investigations revealed that acetate kinase, butyrate kinase, and acetoacetate decarboxylase activities decreased sooner in the mutants than in the parent strain.     

Genetic improvement of technological characteristics of starters for fermented milk products

Possibility for improvement of technological characteristics of lactobacilli using mutations of resistance to rifampicin (rif ^r ) and streptomycin (str ^r ) was studied. Using starter model of Narine Lactobacillus acidophilus INMIA-9602 Armenian diet milk product, it was showed that a possibility for selecting strains with increased rate of milk fermentation and acid production is higher in Rif^r and Str^r mutants induced by nitrosoguanidine than in cultures sensitive to antibiotics. The milk products obtained using Rif^r and Str^r strains had high viscosity, improved texture, increased amount of alive cells and good organoleptic features.     

Mutation and DNA replication in Escherichia coli treated with low concentrations of N-methyl-N′-nitro-N-nitrosoguanidine

N-Methyl-N′-nitro-N-nitrosoguanidine (nitrosoguanidine) causes an unexpectedly high frequency of closely linked double mutants because of its specificity for chromosome regions in replication. Low nitrosoguanidine concentrations (1 μg/ml) in liquid cultures allow replication at the normal rate and are mutagenic. It was expected that mutations would be spread over the chromosome as it replicated, but a high frequency of closely linked double mutants was found.If a thymine auxotroph is grown in the presence of 5-bromodeoxyuridine (BUdR) and nitrosoguanidine, then exposed to 313-nm radiation (which destroys BUdR-substituted DNA), the mutation frequency is much higher among survivors than among non-irradiated cells. It is concluded that nitrosoguanidine inhibits DNA replication in a small fraction of the population and that mutations are induced in that same fraction.Nitrosoguanidine treatment leads to a high frequency of closely linked double mutants under all known conditions.     

Virus-like Particles in USTILAGO MAYDIS: Mutants with Partial Genomes

Mutants with partial genomes for the virus-like particles of U. maydis were recovered following treatment with nitrosoguanidine. Examination of the properties retained by progeny of genetic crosses indicates that the 2.9 x 10⁶ dalton component of double-stranded RNA contains the information for capsid formation and dsRNA replication. Other components appear to contain the information for killer function and immunity to killer. The use of such mutants for studies on the evolution of viruses with segmented genomes is discussed.     

Genetic studies of the ribosomal proteins in Escherichia. coli. II. Altered 30s ribosomal protein component specific to spectinomycin resistant mutants

Summary Spectinomycin resistant (spc ^r) mutants were obtained by treating the cells of E. coli K12, W3637 with nitrosoguanidine. The compositions of ribosomal proteins were analyzed for six out of eleven such spc ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 30s ribosomal subunit from all of the spc ^r-mutants was found to contain the altered 30-4 protein component, while no difference was detected in 50s ribosomal proteins between spc ^r and spc ^s bacteria.Abbreviations used CMC carboxymethyl cellulose - str streptomycin - spc spectinomycin     

A Mutational Study of the Nitrogen-fixing Blue-Green Alga Anabaena doliolum

A heterocystous, non-nitrogen-fixing mutant of the nitrogen-fixingblue-green alga Anabaena doliolum has been isolated followingtreatment with nitrosoguanidine and UV radiation. Some reversiblevariations in the habit and morphology of the alga were inducedfollowing its treatment with nitrosoguanidine.     

Genetic variability in the European minnow,Phoxinus phoxinus (L.)

An electrophoretic study of genetic variation at 11 loci was performedfor a population of European minnows, Phoxinus phoxinus (L.). Ten loci, EST-1 ^*, EST-2 ^* EST-3 ^*,GPD-1 ^*,GPD-2 ^*,GPI-1 ^*,GPI-2 ^*,MPI ^*,6PGD ^* and PGM ^* were polymorphic. IDH ^*wasmonomorphic. The mean number of heterozygotic loci over all 176 fish was 3.05 ± 0.104(SE). Observed mean heterozygosity was 0.28±0.058(SE) and expected mean heterozygosity was 0.27±0.054(SE). EST-2 ^*, EST-3 ^* andPGM ^* were not in Hardy-Weinberg equilibrium. Length,condition, parasite numbers or male breeding characters, i.e. red colorationand tubercles, were not influenced by single enzyme loci.