Genetic analysis of purple adenine (ad-3) mutants induced by methyl methanesulfonate in Neurospora crassa

The mutagenicity of methyl methanesulfonate (MMS) was studied in a genetically marked two-component heterokaryon of Neurospora crassa. Types of genetic alterations detectable in this system are (I) point mutations in the ad-3A and ad-3B loci; (2) multilocus (chromosome) deletions in the ad-3 region, and (3) recessive lethal mutations in the whole genome. Study of the inactivation kinetics of the heterokaryotic and homokaryotic conidial fractions has made it possible to distinguish between nuclear and cytoplasmic inactivation.Forward mutations in the ad-3 region induced by MMS in the heterokaryotic fraction of conidia were obtained by a direct method with the following results: (I) The overall ad-3 forward mutation frequency increases in proportion to the 1.91 power of the concentration of MMS. (2) The forward mutation frequency of point mutations at the ad-3A and ad-3B loci increases in proportion to the 1.68 power of the concentration. (3) The forward mutation frequency of chromosome deletions in the ad-3 region increases more than exponentially with increasing concentrations of MMS. (4) After treatment for 300 min with 20 mM MMS, 15.5% of the ad-3 mutations are multilocus deletions. Tests for genotype and allelic complementation of the point mutations showed that (I) the ratio between ad-3B and ad-3A mutants was 1.75, (2) 52.1% of the ad-3B mutants showed allelic complementation, with 39.2% non-polarized and 12.9% polarized complementation patterns and 47.9% noncomplementing mutants, and (3) both the ratio between point mutations in the ad-3A and ad-3B loci and the spectrum of complementation patterns among the ad-3B mutants were independent of MMS concentration.     

Genetic characterization of ad-3 mutants induced by chemical carcinogens, I-phenyl-3-monomethyltriazene and I-phenyl-3,3-dimethyltriazene, in Neurospora crassa

180 ad-3 mutants of Neurospora crassa induced by 1-phenyl-3-monomethyl-triazene (PMMT) and 56 ad-3 mutants induced by 1-phenyl-3,3-dimethyltriazene (PDMT) were characterized by dikaryon, trikaryon and complementation tests. Results show that the spectrum of genetic alterations induced by PMMT is different from that of PDMT. This suggests that enzymatic dealkylation of PDMT to PMMT does not occur within Neuropsora crassa conidia, and that the mechanism of mutation induction of PDMT in N. crassa is different from that of PMMT. Hydrolytic breakdown products or its intact molecule or some other converted forms might be responsible for the mutagenic activity of PDMT.Mutation induction of PMMT in N. crassa appears to be via alkylation of DNA by carbonium ions produced by this compound, the same mechanism proposed for its carcinogenic activity. The frequencies of leakiness, allelic complementation and nonpolarized complementation patterns among PMMT-induced ad-3 mutants are similar to those of ad-3 mutants induced by other potent chemical carcinogens, such as MNNG and the aflatoxins.     

Metabolic activation of dimethylnitrosamine and diethyl-nitrosamine to mutagens

Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) are not mutagenic by themselves, but they can be converted by mammalian enzymes to highly mutagenic products. As indicators for mutagenic activity, Neurospora crassa and Salmonella typhimurium were used. The ad-3 forward-mutation system was used to detect specific locus mutations; mutants in this system can range from multi-locus deletions to leaky mutations. The induction of mutations in S. typhimurium is detected as induction of histidine revertants of the histidine-requiring strain G₄6. The activation of DMN is microsomal, inhibited by SKF 525-A, and requires co-factors. The activating enzyme is induced in mice by pretreatment with phenobarbital, 3-methylcholanthrene and butylated hydroxytoluene. The mutagenic activity of the reaction products is directly correlated with the metabolic formation of formaldehyde with and without induction by 3-methylcholanthrene and across strains of mine. Formaldehyde does not contribute to the mutagenic activity of the reaction products. It is clear from the data that the reversion sites in G₄6 are more sensitive than the ad-3 loci of Neurospora crassa to the mutagenic action of DMN metabolites formed by mammalian liver. The microsomal assay is a few orders of magnitude more sensitive than the intraperitoneal host-mediated assay, and the intrahepatic host-mediated assay is a few orders of magnitude more sensitive than the in vitro microsomal system.     

Allelspezifische suppressormutationen vonSchizosaccharomyces pombe

By comparing the intragenic distribution of suppressor sensitive mutants in fine structure maps, 13 allele specific suppressor mutations (isolated from revertants in adenine dependent mutants of constitutionad ₇) have been analyzed for their allele specific patterns of action in three different groups of mutants blocked in adenine biosynthesis. The 13 suppressor mutations, which have resulted from mutations at seven different suppressor loci, are characterized by four different suppression patterns. Three of these patterns, which partially overlap, are not locus specific since they include sensitive mutants at each of the three lociad ₇, ad₆ andad ₁ studied. The relative frequency of mutants sensitive to one or the other of the suppressors of this type, the absence of osmotic-remedial strains among the suppressor sensitive mutants, and the polarized complementation behaviour of one suppressiblead ₆ mutant and two suppressiblead ₁ mutants capable of interallelic complementation, suggest that the suppression mechanism involves misreading of a mutant triplet of the nonsense type.     

Mutation Induction by Difunctional Alkylating Agents in NEUROSPORA CRASSA

The genetic characterization of ad-3 mutants of Neurospora crassa induced by two carcinogenic difunctional akylating agents, 1,2,4,5-diepoxypentane (DEP) and 1,2,7,8-diepoxyoctane (DEO), has shown that point mutations at the ad-3B locus have similar complementation patterns. In addition to the induction of point mutations, DEP induces a low frequency (7.5%) of multilocus deletions, whereas DEO induces an extremely high frequency (42.0%). The distribution of the different classes of ad-3 mutants and the frequency of multilocus deletion mutants among DEP-induced mutants are not significantly different from those induced by the monofunctional alkylating agents EI, EMS and ICR-177 at comparable forward-mutation frequencies. Moreover, the frequencies of DEP-induced ad-3B mutants showing allelic completion or having nonpolarized complementation patterns are similar to those of ad-3B mutants induced by monofunctional agents. It is suggested, therefore, that the mechanism of mutation-induction by DEP in N. crassa is similar to that of monofunctional alkylating agents. Mutation-induction by DEO probably results both from the mechanism of action of monofunctional alkylating agents and from inter-strand cross-linkage of the DNA molecular by the two functional epoxy groups.     

A genetic and functional characterization of adenine mutants induced in yeast by 1-nitroso-imidazolidone-2 and nitrous acid

Summary The mutational activity of NIL and HNO₂ is compared in a forward mutation system of Saccharomyces cerevisiae involving ad₁ and ad₂ loci. The comparison includes the distribution of ad₁ to ad₂ mutations as well as certain functional aspects, namely, the frequency of KCl remedials and intragenic complementation with the tester strains. The results show that NIL and HNO₂ mutants are similar both in the distribution of ad₁ to ad₂ and in their functional aspects. Our findings suggest, furthermore, that in a certain group of mutants—mutants complementing with the tester strains—complementation is incompatible with KCl repair.     

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.     

Hydroxylamine-induced purple adenine (ad-3) mutants in Neurospora crassa. I. Characterization of mutants by genetic tests

The genetic effects of hydroxylamine (HA) on Neurospora crassa were studied in an effort to understand the difference between the results obtained on very simple prokaryotic systems and those obtained with mammalian systems. A 2-component heterokaryon was used to study the inactivation of conidia and the induction of recessive lethal mutations at specific loci and over the entire genome. The heterokaryon is heterozygous for 2 closely linked loci, ad-₃A and ad-₃B, in the ad-₃ region. Specific locus mutations can result from either point mutation or chromosome deletion. The results were as follows: (1) Both homokaryotic and heterokaryotic conidia had multi-hit survival curves, and there was no difference between the survival levels of the two as a function of treatment time. (2) The frequency of recessive lethal mutations in the ad-₃ region increased as the square of treatment time.     

Endogenous purine metabolism in the conidia of wild type and certain adenine mutants of neurospora crassa I. The nature of the reserve pools and pool utilization during adenine starvation

Conidia of four adenine auxotrophs (ad 9, ad 3B, ad 8 and ad 4 of Neurospora crassa differ in their ability to germinate on adenine-deficient medium. A large percentage of the ad 9 and ad 3B mutant conidia germinate while those of ad 8 and ad 4 mutant do not. No correlation was found between the size of the conidial purine reserves and the conidial ability to germinate. In all the strains the major fraction of the conidial purine reserved pools was inosine. The ad 8 and ad 4 mutants are blocked after IMP formation in the adenine biosynthetic pathway and therefore cannot use the stored inosine for germination. Pool-utilization studies indicated that in all strains investigated some of the purine reserved were lost from the conidia during incubation. In the most readily germinating strain, ad 9, only small amounts of the purine pool were lost from the conidia and a large portion of the reserve pool was used for nucleic acid synthesis. The nature of the purine reserves present in the conidia, and the ability of the strains to prevent loss of the stored purines from the conidia appear to be among the factors influencing the conidial germination of the adenine mutants of N. crassa.     

Mild RIP — an alternative method for in vivo mutagenesis of thealbino-3 gene inNeurospora crassa

We have used a biological phenomenon that occurs inNeurospora crassa, termed Repeat-Induced Point mutation (RIP), to create partially functional mutant alleles of thealbino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. A total of 70 RIP-inducedal- 3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid biosynthesis. Nucleotide sequence analysis of theal-3 gene in five of the RIP-induced mutants revealed that in each case RIP had introduced no more than six point mutations. The low frequency of RIP mutants (0.42%) and the isolation of only leaky mutants with very few mutations suggest that ascospores containing a heavily mutatedal-3 gene do not survive. These results are evidence that the RIP phenomenon, used to inactivate and silence duplicated genes inN. crassa, may be exploited in its mild version as a method of sequence-specific in vivo mutagenesis to obtain functional mutant alleles ofNeurospora genes. This mild form of mutagenesis may be particularly advantageous in selecting for leaky mutations in essentialNeurospora genes.     

Genetic and Morphological Study of Aggregation in the Cellular Slime Mold POLYSPHONDYLIUM VIOLACEUM

A system for genetic analysis in the cellular slime mold P. violaceum has been developed. Two growth-temperature-sensitive mutants were isolated in a haploid strain and used to select rare diploid heterozygotes arising by spontaneous fusion of the haploid cells. A recessive mutations to cycloheximide resistance in one strain enables selection of segregants, which often appear to be aneuploid.—Aggregation-defective (ag^- ) mutants having a wide range of phenotypes were isolated in both temperature-sensitive strains after nitrosoguanidine treatment, and complementation tests were performed between pairs of these mutants. Of 380 diploids isolated, 32 showed defective aggregation and were considered to contain 2 noncomplementing ag^- mutations. Among noncomplementing mutants interallelic complementation is common. Noncomplementing mutants fall into 4 complementation groups, and those within each complementation group are phenotypically similar. Statistical analysis of the results suggests that the number of complementation units involved in aggregation is about 50.     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress.     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.     

Mutations Affecting Synthesis of β-Galactosidase Activity in the Yeast KLUYVEROMYCES LACTIS

Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac^-) were isolated and characterized. All of the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9. Strains bearing mutations in lac3, lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal^-). Since the Gal^- and Lac^- phenotype co-segregate, they are probably due to a single mutation. Strains bearing mutations in any of the seven complementation groups grow normally on glucose. However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium. Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose. Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate chromosome 2. The remaining five groups are unlinked. Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.——To further characterize the nature of the Lac^- phenotype, the basal and inducible level of β-galactosidase activity were measured. All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels. Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups. In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells. Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of β-galactosidase. In summary, these data indicate that several genes are necessary for synthesis of β-galactosidase activity.     

Genetic recombination in Escherichia coli. IV. Isolation and characterization of recombination-deficiency mutants of Escherichia coli K12

19 independent recombination-deficient mutants were isolated. 7 carried mutations that mapped near or in the recB and recC genes between thyA and argA. 10 mutants carried mutations cotransducible with pheA and exhibited no complementation with recA in temporary zygotic diploids.     

Complementation Analysis of Metabolite-Resistant Mutations with Forced Heterokaryons of NEUROSPORA CRASSA

The double mutant strain pyr-3 arg-12^s is a prototroph because a common precursor of arginine and pyrimidine is supplied by the arginine pathway. Growth of this strain is inhibited by exogenous citrulline or arginine. Citrulline-resistant mutants of this strain were selected, and they resulted from modifier mutations at other loci. Forced heterokaryons were used to study complementation among these modifiers. Since the complementation test requires the scoring of non-growth as the positive result, there was concern that variations in nuclear ratios could give erroneous results. This possibility does not seem significant, since groups of mutants established by complementation correspond with groups established by physiological, enzymatic, and recombinational measurements.—The technique has revealed that the most frequently mutated loci are arg-1 and what is probably un-3. Arg-1 mutations affect the conversion of citrulline to argininosuccinate, while un-3 mutations reduce the citrulline uptake rate. Since most of these mutations are of the intracistronic complementing type, a complementation map was constructed for most of the affected loci. The high proportion of complementors in each map can be explained by assuming that partially functioning gene products are more likely to complement with each other than are those which are nonfunctional.