nitro-N-nitrosoguanidine Research Articles

Basidiocarp development inCoprinus macrorhizus

The dikaryon #5026 (A2B2)+#5132 (A7B7) of the basidiomyceteCoprinus macrorhizus was treated with ultraviolet light (UV),N-methyl-N′-nitro-N-nitrosoguanidine (NG), and 5-bromouracil (BU) in order to induced developmental variations in fruiting. Among a total of 11,383 isolates, 742 were homokaryons. Fruiting test was made with the remaining 10,641 dikaryotic isolates, of which 1,594 showed abnormality in basidiocarp development. Both UV and NG were very efficient in inducing such variations, but BU was not. The induced variations were classified into seven basic types as follows: 1) “knotless”, in which hyphal knots, the first sign of fruiting, do not differentiate; 2) “primordiumless,” in which hyphal knots are formed, but no further development occurs; 3) “maturationles”, in which primordia are formed, but the maturation of primordia into adult fruit bodies does not occur; 4) “elongationless”, in which the elongation of stipe is blocked, and a basidiocarp with a very short stipe is produced; 5) “expansionless,” in which pilei do not expand normally; 6) “sporeless,” in which the formation of basidiospores is blocked and albinic pilei bearing none or only a small amount of spores are produced; and 7) “autolysisless,” in which the autolysis of pilei does not appear to occur. It has been noticed that the four steps of maturation, i.e. stipe elongation, pileus expansion, spore formation, and perhaps pileus autolysis, can proceed independently. Compound types of variations such as “elongation-expansionless,” “elongation-sporeless,” “expansion-sporeless,” and “elongation-expansion-sporeless” were also induced. UV treatment induced maturationless at the highest rate, while NG treatment sporeless.

Induction of Cycloheximide-Resistant Mutants in Saccharomyces cerevisiae with N -Methyl- N ′-Nitro- N -Nitrosoguanidine and ICR-170

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cycloheximide-resistant mutations in Saccharomyces cerevisiae, but few, if any, resistant mutants are induced by the acridine mustard ICR-170. Cycloheximide sensitivity in yeast is associated with the ribosome, and treatment with the antibiotic at concentrations of 2 mug/ml results in complete inhibition of protein synthesis. Missense mutations induced by MNNG probably lead to the loss of cycloheximide binding sites on the ribosome, resulting in resistance to the antibiotic without altering the activity of the organelle in protein synthesis. ICR-170, however, induced primarily frameshift mutations that would alter ribosome structural integrity, resulting in cell death rather than resistance. ICR-170 and MNNG are both mutagenic in a system in which base-pair substitution and frameshift mutations can be detected. These results indicate that cycloheximide resistance in S. cerevisiae, like streptomycin and spectinomycin resistance in Escherichia coli, can be induced by base-pair substitution mutagens but not by frameshift mutagens such as ICR-170.

Production of L-Threonine by Analog-resistant Mutants

Mutants resistant to α-amino-β-hydroxyvaleri0c acid (AHV) were derived from various bacteria which belong to Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Bacillus by mutational treatment with N-methyl-N′-nitro-N-nitrosoguanidine(NTG), and screened for their ability to produce l-threonine. A number of l-threonine producers were obtained from each group of bacteria. Among them, the mutants derived from C. glutamicum KY9159(Met−) were further mutagenized with NTG to derive thialysine(S-Lys)-resistant mutants. An AHV-resistant mutant, KY10484 was proved to be much more sensitive to the growth inhibition by thialysine than the parent strain, KY9159. From KY10484, a number of AHV- and thialysine-resistant mutants were derived. Approximately a half of these mutants were found to produce more l-threonine than KY10484. Among these mutants, KY10440 (Met−, AHVR, s-LysR) was used to investigate the cultural conditions for l-threonine production. The growth of KY10440 decreased largely with addition of l-homoserine, a threonine precursor. l-Asparagine, l-cystine, l-glutamine or l-arginine partially reversed the inhibitory effect of l-homoserine. Addition of these amino acids at low level led to increase l-threonine production. The amount of l-threonine accumulation reached to a level of 14mg/ml with a medium containing 10% glucose and to a level of 10 mg/ml with a medium containing 5% molasses (as glucose). Another AHV- and thialysine-resistant mutant, KY10251 which was also derived from KY9159 was found to produce both 9 mg/ml of l-threonine and 5.5 mg/ml of l-lysine in a culture broth.

Monofunctional alkylating agent-induced inactivation, mutagenesis and DNA degradation in an Escherichia coli mutant deficient in DNA polymerase.

The DNA polymerase deficient mutantE. coli P3478polA1 is extremely sensitive to the lethal action of N-methyl-N′-nitro-N-nitrosoguanidine (NG) and methyl-methanesulfonate (MMS). ThepolA1 mutant has an almost unaffected mutability induced by NG or MMS and shows reduced ability to propagate MMS-treated phage T7. NG and MMS induce marked breakdown of DNA and inhibit significantly DNA synthesis in thepolA1 mutant. The obtained results suggest the thepolA1 mutant is unable to repair single-strand breaks of DNA induced by monofunctional alkylating agents. The suggestion is confirmed by the demonstrated sensitivity of thepolA1 mutant to thymine starvation (TS).

Experimental production of gastric cancer by oral administration of N-Methyl-N′-Nitro-N-Nitrosoguanidine (NG) and gastric secretion during carcinogenesis of stomach

Experimental production of gastric cancer by oral administration of N-Methyl-N′-Nitro-N-Nitrosoguanidine (NG) and gastric secretion during carcinogenesis of stomach

Induction of mutants of Staphylococcus aureus 100 with increased ability to produce enterotoxin A.

As a result of serial exposures to a mutagenic agent, N-methyl-N'-nitro-N-nitrosoguanidine, the yield of enterotoxin A produced by the last mutant in the series was increased nearly 20-fold over the amount produced by the parent Staphylococcus aureus 100.

Mutant of Escherichia coli K-12 defective in D-glucosamine biosynthesis.

A mutant was isolated from a derivative of Escherichia coli K-12 after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. This mutant contained normal levels of 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (deaminating) (EC 5.3.1.10), but no detectable activity of l-glutamine:d-fructose-6-phosphate amino-transferase (EC 2.6.1.16). It required either N-acetyl-d-glucosamine or d-glucosamine for growth, and went into rapid lysis when the supply of these compounds was exhausted. In medium containing 11% sucrose, the cells were converted into spheroplasts in the absence of d-glucosamine.

Temperature Dependency of the Mutagenic Action of

The effectiveness of N-methyl-N'-nitro-N-nitrosoguanidine (NG) in inducing mutations in Streptomyces cacaoi was demonstrated with two systems: reversion to prototrophy of an isoleucine-requiring mutant and the induction of forward mutation of a prototrophic strain. An optimal condition for yielding a high frequency mutation was set as follows: Treat-ment of spores with 1mg/ml of NG in 0.016M phosphate buffer, pH 6, at 42°C for 60min. In Streptmyces cacaoi, the mutagenesis depended highly on the temperature of NG treatment; elevation of the treatment temperature resulted higher frequency of mutation. Only a little dependency on temperature was shown in Escherichia coli, but the dependency was also observed when the Streptomyces was treated with ethyl methanesulfonate. The significance of these results for considering mechanism of NG action was discussed.

Effects of some chemical mutagens on survival and reversion of some mutant strains of Fusarium spec.

The effects of acridine orange, malachite green, toluidine blue, oxytetracycline hydro chloride, caffeine, and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) have been investigated in the mutant strains R 79, RM, WR, diauxotroph compact and widespread of Fusarium spec. Only caffeine and, as it is shown for the first time in this organism, also NTG induced reversions in the strains R 79, RM and WR.

Effects of some chemical mutagens on survival and reversion of some mutant strains of Fusarium spec.

AbstractThe effects of acridine orange, malachite green, toluidine blue, oxytetracycline hydro chloride, caffeine, and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (NTG) have been investigated in the mutant strains R 79, RM, WR, diauxotroph compact and widespread of Fusarium spec. Only caffeine and, as it is shown for the first time in this organism, also NTG induced reversions in the strains R 79, RM and WR.

Mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine on Gaffkya tibisci at alkaline pH.

Good yields of mutants were obtained by incubating cells of Gaffkya tibisci with N-methyl-N'-nitro-N-nitrosoguanidine in alkaline buffer. Nearly 50% of the survivors were auxotrophic mutants under the conditions described.

Inactivating and mutagenic effects of nitrosoguanidine on simian virus 40.

N-methyl-N'-nitro-N-nitrosoguanidine (NTG) in high concentrations inactivates extracellular simian virus 40. No mutants were found in stocks of virions partially inactivated by NTG under the experimental conditions employed. In contrast, NTG in low concentrations inhibits the intracellular replication of simian virus 40 and is an effective mutagen of the intracellular virus. Six temperature-sensitive mutants of simian virus 40 have been isolated for genetic studies of the virus and its interaction with mammalian cells.

Reversion of the Frameshift hisD3018 of Salmonella typhimurium

Previous studies on histidinol dehydrogenase from His(+) revertants have shown that the frameshift mutation hisD3018 is a +1 type, resulting from inclusion of an extra cytidylate residue in messenger ribonucleic acid. Histidinol dehydrogenase from newly isolated spontaneous and N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced intragenic revertants has been examined for amino acid replacements. The results provide additional evidence that NG can delete guanine plus cytosine base pairs from deoxyribonucleic acid. One spontaneous revertant was found to result from a +2 addition of approximately 16 nucleotide residues before the +1 parent frameshift, and another by a -4 deletion about six residues before the same. Circumstantial evidence suggests the in vivo codon assignment GAG for glutamic acid. A region of histidinol dehydrogenase highly permissive of amino acid changes encoded in the minus (-) phase is now apparent.

Characteristics of a stable, filamentous mutant of a coccoid blue-green alga.

Filamentous mutants were induced in a coccoid blue-green alga, Agmenellum quadruplicatum strain BG1, after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG). The mutants fall into two general classes: filaments with cross walls and filaments without cross walls. All mutants of these general types derived from BG1 are stable and have growth rates the same as or very similar to the wild type under a variety of conditions. Detailed examination of one mutant, 53SB2, revealed no difference in deoxyribonucleic acid content nor in base ratios. Mutant 53SB2 did not revert to the normal cell size and shape when grown under different physical conditions nor upon the addition of potential reversing agents to the basal medium. It is our general experience that filamentous mutants such as those described here in BG1 are commonly induced in other coccoid blue-green algae after NTG treatment.

Isolation of L-forms of Bacillus subtilis which grow in liquid medium.

L-forms of Bacillus subtilis can be isolated by treatment of the parent strain sequentially with N-methyl-N'-nitro-N-nitrosoguanidine and lysozyme and selection of the surviving protoplasts on semisolid medium containing 2,000 units of penicillin per ml. Some of these clones can be adapted to grow in liquid cultures containing 1.2 m NaCl. This method will aid in the isolation of cell wall mutants which require hypertonic medium for growth.

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Morphological changes were studied in the glandular stomach of rats with carcinogenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine (NG). NG was given to 100 rats in their drinking water at the level of 167 μg/ml for 40 weeks and then 84 μg/ml until the end of the experiment. Rats were killed periodically and histological changes in the glandular stomach were examined. Three types of glandular proliferation were distinguished: regenerative glandular hyperplasia, adenomatous hyperplasia, and adenocarcinoma. These lesions were mainly in the antrat region of the stomach. After atrophy and erosion of the mucosa, regenerative glandular hyperplasia, with irregular glands at the margins of erosions, developed in the 3d–5th week, and these lesions persisted for the rest of the experiment. Adenomatous hyperplasia, characterized by excessive glandular proliferation with scanty cellular atypism, developed in the 20th week, and these lesions were found in the middle of the experimental period. Adenocarcinoma, consisting of excessive glandular proliferation with cellular atypism, appeared in the 30th week and gradually invaded to the submucosa, the muscularis propria, and the serosa. After the 60th week, almost all rats had adenocarcinoma with serosal invasion.

Comparative ultrastructure of a filamentous mutant and the wild type ofAgmenellum quadruplicatum

A filamentous variant of the marine coccoid cyanophycean alga,Agmenellum quadruplicatum has been produced after treatment of cells with the chemical mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The mutation to the filamentous condition is stable and has been compared ultrastructurally with the unicellular wild type. The structural basis for the maintenance of the filamentous state is due to a failure of the homogeneously-structured transverse wall to differentiate into the stratified longitudinal wall. A manifestation of this condition is the continued synthesis of the investment membrane resulting in proliferations at each constriction. The wild type cells are nearly spherical while the filamentous mutant cells are smaller and elongate. An analysis of the structure and morphology of the nucleoplasmic regions is presented for both strains and a correlation between the morphology of these regions and the unicellular and filamentous conditions is made.

Sulfur-deficient transfer ribonucleic acid in a cysteine-requiring, "relaxed" mutant of Escherichia coli.

A cysteine-requiring mutant of the parent strain Escherichia coli Hfr Cavalli (RC(rel), Met(-), lambda) has been isolated. The mutant was selected by using replica plating after mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine. The mutation appears to be in the gene for sulfite reductase, since the mutant could utilize sulfide but not sulfite as a sulfur source. The mutant was found to be RC(rel) with respect to both methionine and cysteine. During cysteine starvation, transfer ribonucleic acid (tRNA) deficient in 4-thiouracil was produced, and in vivo studies indicate that this tRNA can accept sulfur groups to a greater extent than normal tRNA. Further, there were differences both in the rate and extent of amino acid acceptance between normal and sulfur-deficient tRNA. This suggests that thionucleotides are involved in at least one of the biological functions of the tRNA molecule.

Nature of the hisD3018 Frameshift Mutation in Salmonella typhimurium

Histidinol dehydrogenase from three differing revertants of ICR-191A-induced frameshift hisD3018 has been purified and examined for amino acid replacements. The enzyme from one spontaneously arising revertant, R7, contains an extra proline residue, whereas that from another, R5, contains an extensive frameshifted sequence, four amino acid residues of which have been identified to date. The amino acid replacement data are in agreement with the in vitro code word assignments and allow the characterization of the hisD3018 frameshift as an addition of one nucleotide pair, most likely guanine plus cytosine. Enzymatic data for those ICR-191A-induced revertants of hisD3018 arising within the hisD gene indicate that the enzyme is wild type and, therefore, that ICR-191A can cause deletions as well as additions of single base pairs. The wild-type amino acid sequence is restored in enzyme from an N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced revertant, R29, suggesting that NG is a base-deleting as well as a base-substituting mutagen. The unusual response of hisD3018 to external suppressors is considered in terms of reinitiation of protein synthesis out of phase, coupled with suppression of a nonpermissive missense codon so generated, and of an alternative hypothesis invoking a true frameshift suppressor transfer ribonucleic acid with an extended or deleted anticodon.

Externally suppressible frameshift mutant of Salmonella typhimurium.

Prototrophic revertants of ICR-191A-induced frameshift mutant hisD3018 have been induced spontaneously by ICR-191A and N-methyl-N'-nitro-N-nitrosoguanidine (NG) treatment. In each case two genetically distinct prototroph classes were differentiated by transducibility into his deletion recipients: (i) transducible, generally fast-growing revertants within the hisD gene producing from 10 to 100% of normal amounts of histidinol dehydrogenase and (ii) nontransducible slow-growing prototrophs with very low levels of enzyme activity of which at least some arose by external suppression. These nontransducible revertants, whether arising spontaneously or in the presence of ICR-191A or NG, contain histidinol dehydrogenase which is electrophoretically similar to the wild-type enzyme.