Genetic and biochemical properties of thialysine-resistant mutants of Saccharomyces cerevisiae

Three groups of lysine-excreting, thialysine-resistant mutants of Saccharomyces cerevisiae were derived from the wild-type strain (X2180) by mutagenic treatment and selected on the basis of a cross-feeding assay. Mutants MNNG2-9, MNNG2-27, MNNG2-39 and MNNG2-62 (group 1) exhibited a 2:2 segregation for thialysine resistance following mating with a wild-type strain and a lower than wild-type lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was dominant in specific hybrids. Mutant MNNG2-2 (group II) was similar to group I mutants except that the thialysine-resistant phenotype was recessive in the hybrid. Mutant MNNG3-142 (group III) exhibited an irregular ratio of segregation of thialysine resistance and a significantly lower lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was recessive in the hybrid. The growth of both group I and group III mutants was temperature-sensitive. The thialysine-resistant mutants exhibited pleiotropic properties including the increased production and excretion of lysine, thermosensitive growth and an impairment of lysyl-tRNA synthetase activity.     

Effects of hydroxylamine and N-Methyl-N′-Nitro-N-nitrosoguanidine in Mimulus cardinalis (Scrophulariaceae): Survival curves and dominant mutants

Summary The lethal and mutagenic effects of hydroxylamine (HA) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) were investigated in the higher plant Mimulus cardinalis. MNNG was found to be more toxic than HA. The shapes of the survival curves obtained at different concentrations of HA and MNNG are interpreted on the basis of decreased biological activity of the solution to increased age of solution. Based on the appearance of chlorophyll-deficient mutants, MNNG is mutagenic in Mimulus. No albinos were detected in HA treated plants. A total of 67 putative mutants were isolated in the mutation spectra of HA and MNNG treated plants. The frequency of mutants induced by HA and MNNG are different. MNNG is mutagenic at 1/10 the concentration of HA in inducing putative mutations in M ₁ plants.A portion of this work will be submitted by the senior author to the Faculty of Miami University in partial fulfillment of the Doctor of Philosophy degree.     

Ultrastructural organization of secondary mutants ofEuglena gracilis induced by N-methyl-N′-nitro-N-nitrosoguanidine

Photosynthetic mutants ofEuglena gracilis obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or nitrovin treatment were treated secondarily with MNNG and bleached mutants were isolated. In all mutant strains maintained in the light-grown condition some remnants of plastids were always observed. However, there are differences in the internal organization of secondary mutants between samples taken for examination after 15 and 200 generations of subcultivation. On the other hand, there are no differences in the internal organization of secondary mutants derived from primary photosynthetic mutants and bleached mutants induced directly from wild-type euglenas by MNNG treatment.     

Isolation and characterization of respiration-deficient mutants inAspergillus ochraceus

Two respiration-deficient mutants (rd) were isolated from the acetate-nonutilizing mutants (acu) induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) inAspergillus ochraceus. A complementation analysis of the tword mutants indicated that MNNG had caused a mutation at a single locus. The diameter of the tword mutant colonies in glucose medium was found to be small in comparison to that of the wild type and the otheracu mutants; the diameter of the isolated mutant colonies in acetate medium was very small. The grown zone ofrd mutants remained colorless up to 20 h incubation in 2,3,5-triphenyltetrazolium-overlaid solid Czapek-Dox medium and it turned pink after prolonged incubation, whereas the wild type and the otheracu mutants became pink within 30 min in the same medium. Therd mutants were further characterized by measuring the respiratory activities of intact mycelia in the presence of glucose.     

Genetic effects of N-methyl-N''-nitro-N-nitrosoguanidine in Neurospora crassa

Summary The mutagenicity of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was studied with a genetically marked, balanced heterokaryon of Neurospora crassa. Types of genetic alterations detectable in this system are (a) point mutations in the ad-3 A and ad-3 B loci, (b) multilocus (chromosome) deletions in the ad-3 region, and (c) recessive lethal mutations in the whole genome. Study of the inactivation kinetics of the heterokaryotic and homokaryotic conidial fractions makes it possible to distinguish between nuclear and cytoplasmic inactivation.Forward mutations in the ad-3 region induced by MNNG in the heterokaryotic fraction of conidia were obtained by a direct method, with the following results: (1) forward-mutation frequency increases as the square of the time of treatment, (2) MNNG is an extremely efficient mutagen, e. g., the frequency of mutation in the ad-3 region (2 loci) was 0.14% after 240 min treatment with 25 M MNNG at pH 7.0 with 73.4% survival, (3) at least 98.1% of the MNNG-induced ad-3 mutants are point mutations, (4) tests for genotype and allelic complementation showed that (a) the frequency of genotypes was ad-3 A=19.7%, ad-3 B=80.3% and ad-3 A ad-3 B=0.0%, and (b) 81.8% of the ad-3 B mutants have allelic complementation with 79.9% nonpolarized and 1.9% polarized complementation patterns and 18.2% noncomplementing mutants, and (5) the ration between mutations in the ad-3 A and ad-3 B loci and spectrum of complementation patterns among the ad-3 B mutants was independent of dose. Comparison of the spectrum of the complementation patterns among ad-3 B mutants induced by MNNG with the spectrum among ad-3 B mutants induced by 2-aminopurine, nitrous acid, hydroxylamine, and the acridine mustard derivative ICR-170 suggests that the majority of the MNNG-induced mutants have guanine-cytosine at the mutant site.Abbreviations used in this paper GC guanine-cytosine - MNNG N-methyl-N-nitro-N-nitrosoguanidine - HA hydroxylamine - AT adenine-thymine - MMS methyl methanesulfonate - 2AP 2-aminopurine - ICR-170 acridine mustard derivative-(2-methoxy-6-chloro-9-[(ethyl-2-chloroethyl) amino propylamino] acridine dihydrochloride) - NA nitrous acid Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

Random mutagenesis and use of 2-deoxy-D-glucose as an antimetabolite for selection of α-amylase-overproducing mutants of Aspergillus oryzae

By using 2-deoxy-D-glucose, selection of different mutants of Aspergillus oryzae PTCC 5164, which were produced by random mutagenesis by u.v. radiation, nitrous acid and N-methyl-N-nitro-N-nitrosoguanidine (MNNG), was studied. 2-Deoxy-D-glucose, a well-known antimetabolite, was used to isolate derepressed mutants. The mutational and lethal effects of these mutagens on conidia of A. oryzae were compared and the frequency distribution of isolated mutants, in the presence of 2-deoxy-D-glucose, was determined. Potent mutants, which produced higher dextrinizing and saccharogenic activities, were isolated. The best strain was a result of mutagenesis by nitrous acid, which produced 6.73 times more dextrinizing and 5.13 times more saccharogenic activity than the parent strain. In general, the mutants obtained by nitrous acid and u.v. were more potent than those obtained by MNNG.     

Induction of Mendelian and non-Mendelian streptomycin resistant mutants during the synchronous cell cycle of Chlamydomonas reinhardtii

Summary In synchronized cultures of Chlamydomonas reinhardtii N-methyl-N-nitro-N-nitrosoguanidine (MNNG) was found to selectively mutate replicating forms of nuclear DNA. This conclusion was based on the 15- to 30-fold increase in the recovery of MNNG induced Mendelian streptomycin resistant mutants (sr-1) which was correlated with mutagenesis during the period of nuclear DNA replication. No concomitant increase in the recovery of non-Mendelian streptomycin resistant mutants (sr-2) occurred during this same period.Mutagenesis at the time of chloroplast DNA replication, however, resulted in a 1.5- to 1.6-fold increase in the recovery of both sr-1 and sr-2 induced mutants.     

Protective effect of ascorbic acid on bleaching activity of N-methyl-N′-nitro-N-nitrosoguanidine and furazolidone

Exposure of autotrophic cells ofEuglena gracilis to MNNG and furazolidone results in an irreversible loss of chloroplasts leading to a production of white mutants (permanent bleaching). Induction of the white mutants is significantly decreased by ascorbic acid, especially during the first stages of this mutation process.     

Gene expression in E. coli after treatment with streptozotocin

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.     

Lysine Overproduction Mutations in the YeastSaccharomyces cerevisiae Are Introduced into Industrial Yeast Strains

Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature [1]. A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants. The intracellular content of lysine was also increased by 30%. The genetic nature of lysine overproduction was studied in this strain. An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1 ^R) and the other is involved in the regulation of lysine production (PRL). Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome. Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses. The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.     

Isolation and characterization ofPhaffia rhodozyma mutants

Mutagenic treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) inPhaffia rhodozyma generated 15 mutants with a wide diversity of color variants ranging from white to dark red. Characterization of the mutants by absorption spectra, TLC and HPLC was performed. Two categories could be distinguished: astaxanthin hyperproducing and astaxanthin hypoproducing mutants. Hyperproducing mutants exhibited considerable increases in astaxanthin content whereas hypoproducing mutants showed higher β-carotene contents than the wild-type strain. The characterization of carotenoid mutants inP. rhodozyma could contribute to the knowledge of the biosynthetic pathway of astaxanthin production of this microorganism.     

Isolation and characterization of pigment mutants from a filamentous cyanobacterium

Five strains of a pigment mutant were isolated following UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis from a non-nitrogen fixing mutant of the cyanobacteriumGloeotrichia ghosei. Two of them (B-1 and V-1) were isolated by UV mutagenesis and other three (B-3, B-7 and Br-6) by MNNG mutagenesis. Among the five strains cultures of three strains (B-1, B-3 and B-7) were typically blue-green in colour. Culture of strain V-1 was found to be violet-pink and of Br-6 was brownish in colour. The parent strain of these mutants was dark-blue in colour. Blue-green mutants showed the predominance of phycocyanin (610 nm) whereas violet-pink and brown strains showed the predominance of phycoerythrin (550 nm) in the absorption spectra of water-soluble pigments. In contrast to these strains their parent strain showed both the absorption peaks (at 550 and 610 nm). Occurrence of stable pigment mutants of a filamentous cyanobacterium indicates that the synthesis of water-soluble pigments is genetically controlled in these mutant strains.     

L-lysine production at 65°C by auxotrophic-regulatory mutants ofBacillus stearothermophilus

Summary The amino acid L-lysine was produced from auxotrophic-regulatory mutants ofBacillus stearothermophilus at a temperature of 60–65°C. One of the mutants (AEC 12 A5, S-(2-aminoethyl)-cysteine^r, homoserine^–), produced L-lysine at the concentration of 7.5 g/l in shaken flasks in minimal medium containing 5% glucose. Culture conditions for optimizing L-lysine production were not investigated. The aspartokinase activity of the wild strainB. stearothermophilus Zu 183 was inhibited by lysine alone and by threonine plus lysine. AEC resistant mutants showed an aspartokinase activity genetically desensitized to the feedback inhibition. Optimal temperature and pH of aspartokinase were 45°C and 9.5, respectively. The data provide significant evidence that mutants of the speciesB. stearothermophilus have a potential value for amino acid production.     

The adaptive response of E.coli to low levels of alkylating agent: the role of polA in killing adaptation.

Summary In an attempt to characterise which gene products may be involved in the repair system induced in E. coli by growth on low levels of alkylating agent (the adaptive response) we have analysed mutants deficient in other known pathways of DNA repair for the ability to adapt to MNNG. Adaptive resistance to the killing effects of MNNG seems to require a functional DNA polymerase I whereas resistance to the mutagenic effects can occur in polymerase I deficient strains; similarly killing adaptation could not be observed in a dam3 mutant, which was nonetheless able to show mutational adaptation. These results suggest that these two parts of the adaptive response must, at least to some extent, be separable. Both adaptive responses can be seen in the absence of uvrD ⁺ uvrE ⁺-dependent mismatch repair, DNA polymerase II activity, or recF-mediated recombination and they are not affected by decreased levels of adenyl cyclase. The data presented support our earlier conclusion that adaptive resistance to the killing and mutagenic effect of MNNG is the result of previously uncharacterised repair pathways.     

A Mutant of Arabidopsis thaliana lpar;L.) Heynh. with Modified Control of Aspartate Kinase by Threonine

Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grow on 2 m M lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 × RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1and ak2) were found to be unlinked.     

Isolation and characterization of temperature-sensitive mutants ofAnabaena variabilis impaired in nitrogen fixation

Temperature-sensitive (ts) mutants of the cyanobacteriumAnabaena variabilis ATCC 29413 were isolated following mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and post-treatment with metronidazole at 40°C. Of the 8000 clones isolated and tested, six mutants were conditionally lethal at the restrictive temperature (40°C). All the ts mutants exhibited differences in their rates of growth, chlorophyll content, pigment (phycocyanin and/or chlorophyll) ratios, heterocyst frequency, oxygen evolution and nitrogenase activity at the permissive temperature (28°C). A gradual loss of all the above features occurred after a period of 3 d at 40°C, followed by lysis of the cultures. Cessation of nitrogenase activity was found to be different in the different ts mutants. The temperature-sensitive nature of the mutants is suggested to be due to an impairment in iron metabolism since addition of ferric citrate to cultures at 40°C restored the ability to grow, produce heterocysts and fix nitrogen.     

Biochemical characterization of N-methyl N’-nitro-N-nitrosoguanidine-induced cadmium resistant mutants ofAspergillus niger

Two cadmium resistant mutants (Cd₁ and Cd₂) ofAspergillus niger, among the six isolated by mutagenization with N-methyl N’-nitro-N-nitrosoguanidine (MNNG) at pH 6.4 were selected for the study. Analysis of lipid composition of the mutants and the wildtype indicated that total lipid as well as individual lipids of the cadmium resistant mutants were changed as compared with that of the wildtype. The increased activities of metal-lothionein and reduced activities of D-xylose isomerase and L-phenylalanine ammonia lyase in cell free extract of the cadmium resistant mutants suggested that mutants could allow high concentration of cadmium salt as compared with that of the wildtype. The respiratory activity and intracellular as well as extracellular Cd²⁺ concentration of the mutants reflected the high tolerance of the Cd mutants to cadmium ion.     

Facilitation by pyrimidine deoxyribonucleosides and hypoxanthine of mutagenic and cytotoxic effects of monofunctional alkylating agents in Chinese hamster cells.

Hypoxanthine (Hx), thymidine (TdR) and deoxycytidine (CdR), at concentrations of 10(-5) M increased the yield of 8-azaguanine-resistant (AzGr) mutants induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cultured Chinese hamster V79 cells. The cytotoxicity of MNNG was increased 2-fold in the presence of Hx, and more than 10-fold in the presence of TdR. This cytotoxic effect of TdR was abolished by equal concentrations of CdR, which by itself did not increase the cytotoxicity of MNNG. However, the yield of MNNG-induced AzGr colonies was increased 2--10-fold in the presence of both CdR and TdR. The AzGr colonies displayed phenotypes characteristic of hypoxanthine: guaninephosphoribosyltransferase-deficient (HGPRT-) mutants, or indicative of mutant HGPRT with altered substrate affinities. The nucleosides did not affect the growth or expression time of the HGPRT- mutants; the same extent of alkali-labile DNA damage occurred in cells treated with alkylating agents in the presence and absence of TdR and CdR; and the increase in mutation frequency in the presence of these nucleosides occurred not only with MNNG, but also with ethylating agents. Nucleosides treated with MNNG were not mutagenic, and treatment of the cells with TdR and CdR only prior to treatment with MNNG or only during selection with AzG did not increase the induced mutation frequency. Therefore, the interpretation is proposed that CdR, TdR and Hx produce nucleotide-pool imbalances that increase lethal and mutagenic errors of replication of alkylated DNA.     

Protein lysine methylation contributes to modulating the response of sensitive and tolerant Arabidopsis species to cadmium stress

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analysed the importance of protein lysine methylation for plants to cope with cadmium. We analysed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species, Arabidopsis thaliana and A. lyrata, and in three populations of A. halleri with contrasting cadmium accumulation and tolerance traits. We showed that some proteins are differentially methylated at lysine residues in response to Cd and that a few genes coding KMTs are regulated by cadmium. Also, we showed that 9 out of 23 A. thaliana mutants disrupted in KMT genes have a tolerance to cadmium that is significantly different from that of wild-type seedlings. We further characterized two of these mutants, one was knocked out in the calmodulin lysine methyltransferase gene and displayed increased tolerance to cadmium, and the other was interrupted in a KMT gene of unknown function and showed a decreased capacity to cope with cadmium. Together, our results showed that lysine methylation of non-histone proteins is impacted by cadmium and that several methylation events are important for modulating the response of Arabidopsis plants to cadmium stress.