Allelism of methionine-sensitive mutants of Schizosaccharomyces pombe to loci involved in adenine biosynthesis

Methionine-sensitive mutants of Schizosaccharomyces pombe were found to map at 16 loci, minl-minl6, seven of which are allelic to known ode loci involved in adenine biosynthesis. The mutant minl-106 was shown to be located within the ade6 locus by intragenic recombination analysis. Methionine sensitivity of these mutants is reversible by adenine supplementation and their pigmentation is similar to that of the corresponding ade mutants. We conclude, therefore, that these min mutants are incompletely blocked in adenine biosynthesis. These observations further indicate that adenine biosynthesis in S. pombe is inhibited by methionine or a metabolite derived from it. For some other min mutants showing reversibility of methionine sensitivity by leucine or proline, no allelism to known leu or pro loci was detected.


INTRODUCTION
Like auxotrophic mutants, metabolite-sensitive mutants represent a useful tool for discovering and investigating biochemical pathways and their regulation. Among the few reports existing on amino acid sensitive mutants in fungi, two deal with methionine sensitivity. In Aspergillus nidulans, a methionine-sensitive mutant was found to be defective in an enzyme of methionine catabolism (Pieniazek, Stepien & Paszewski, 1973). One of three methionine-sensitive mutants isolated in Saccharomyces cerevisiae, met-i z , has two outstanding properties: (1) Its methionine sensitivity is reversed by adenine supplementation; (2) it was found to be allelic to the ade2 locus, which codes for an enzyme in adenine biosynthesis (Meuris, Lacroute & Slonimski, 1967;Meuris, 1969). This points to a relationship between methionine and adenine biosynthesis. Further evidence for this comes from the observation of Cutts & Rainbow (1950) that wild-type yeasts form a red pigment when grown in the presence of high methionine and suboptimal biotin concentrations. This red pigment also accumulates in mutant strains blocked in two steps of adenine biosynthesis (Smirnov et al. 1967;Dorfman, 1969;Gutz, 1963;Strauss, 1972). In Schizosaccharomyces pombe this relationship between 262 A. STRAUSS methionine and adenine biosynthesis is also indicated by the finding of Clarke (1965) that a leaky ade7 mutant has an increased adenine requirement when grown on medium containing methionine.
In the present study we investigated by genetical means the possibility of a relationship between methionine-sensitivity and adenine biosynthesis in 8. pombe.
(ii) Media Culture media for 8. pombe (YEA, MMA, MEA) were previously described by Gutz et al. (1974). Strains were usually cultured on tubes or plates containing MEA + adenine (50 mg/1). Media for proline-or leucine-requiring strains were additionally supplemented with L-proline (50 mg/1) or L-leucine (50 mg/1), respectively. Amino acids and nucleic acid bases of the highest purity available were used for supplementation.
(iii) Mutant isolation Mutagenization by iV^-methyl-iV^'-nitro-i^-nitrosoguanidine of wild type cells of 8. pombe, 972 h~ or 975 h+, was performed as described elsewhere (Strauss, 1976). Treated cells were plated on unsupplemented MEA and incubated for 6 days at 30 °C. Colonies were then replicated on MMA and MMA + methionine (100 mg/1). After 3 days incubation at 30 °C all clones showing any degree of growth inhibition on MMA + methionine compared to MMA were picked up, reisolated and backcrossed to the wild type for genetical purification and introduction of the opposite mating type.
(iv) Growth and pigmentation For preculturing, cell material of different strains was inoculated as dots on plates with MEA + adenine (50 mg/1) and incubated for 3 days at 30 °C. The resulting cell material was then transferred to various media by replica plating. Growth and pigment formation were examined qualitatively on these media after 2, 3 and 6 days incubation at 30 °C.
(v) Genetical procedures In general, the genetical techniques for S. pombe described by Gutz et al. (1974) were followed. In contrast to studies with auxotrophic strains, wild type recombinants from crosses involving min mutants were selected on MMA + methionine (100 mg/1). Isolated min mutants were genetically classified into loci by the criss-cross technique (Leupold, 1955). The same test was applied for demonstrating allelism between min mutants and ade mutants. Quantitative methods for determination of recombination frequencies (tetrad analysis, free spore analysis) were performed after Gutz et al. (1974). The complementation test was carried out (again on MMA + methionine) according to Leupold (1970).

RESULTS (i) Genetic classification of min mutants
Mutants of S. pombe with wild-type like growth on MMA but with only slight or no growth on MMA + methionine (100 mg/1) were isolated and further analysed. Out of 44 such methionine-sensitive mutants, 34 could be allocated to 16 different loci by means of the criss-cross technique. These newly described loci, called minl-minl6, contained one to four alleles (Table 1). (ii) Further characterization of mutant phenotypes Standard alleles at all min loci were tested to see whether amino acids other than methionine were capable of inhibiting growth on minimal medium. Some amino acids occasionally had a slight inhibitory effect, but in all cases methionine was clearly the most effective inhibitor. Methionine sensitivity of min mutants disappeared partly or fully on YEA -fmethionine (100 mg/1), indicating that some component of yeast extract alleviates the growth inhibition caused by methionine. We therefore examined the possibility that methionine sensitivity on minimal medium could be reversed by additional supplementation with adenine, guanine, uracil or any of the remaining amino acids. As shows Table 1, methionine sensitivity of the standard alleles was in fact reversible by adenine in ten cases (minl,min2, min4, min5, min6, min9, minlO, minll, minl3 and minl4), by proline in one case (min3) and by leucine in two cases (minl2 and minl5).
(iii) Allelism of min mutants to auxotrophic mutants In order to check for allelism, the standard alleles of all min loci showing a reversion effect by adenine were crossed to representative alleles of the ten ade loci known in S. pombe. The outcome of these crosses, performed by the criss-cross technique, suggests allelism in the following seven cases: minl-ade6, min4-adel, min6-ade9, minlO-ade2, minll-ade3, minl3-ade4, minld-adelO. In tetrad analyses of all these crosses only parental ditypes occurred (Table 2), thus confirming the In the case of the allelism minl-ade6, the approximate map position of a min allele was determined at the intragenic level. From crosses of mini-106 with nine ade6 alleles which are dispersed all over the ade6 recombination map as described by Gutz (1963), recombination frequencies were obtained showing that minl-106 is located within the ade6 locus close to ade6-525 (Table 3).
In a similar way, the standard alleles of the min loci showing a reversion effect by proline or leucine were checked for allelism with proline auxotrophic mutants (prol-1 and pro2-l) and leucine auxotrophic mutants . No allelism could be detected.  Complementation reactionf --------+ * Recombination frequencies are expressed in prototrophic recombinant spores/10 6 plated spores and represent mean values from six crosses.
"f + stands for complementation, -stands for no complementation.
(iv) Pigmentation of min and ade mutants Ade6 and ade7 mutants of 8. pombe, grown under limiting concentration of adenine, accumulate a red pigment (Gutz, 1963). The pigment probably consists largely of polymerized AIR (5-aminoimidazole ribonucleotide), an intermediate of purine biosynthesis (Smirnov et al. 1967;Strauss, 1972). Red pigmentation occurred also in mini mutants grown on MMA, but disappeared when adenine was supplied. If conditions were used where wild type cell material was also pigmented, namely under biotin starvation together with high methionine concentrations (Cutts & Rainbow, 1950), three different responses of pigmentation could be observed among ade and min mutants (Table 4): dark red, light red and white. Considering the gene-enzyme relationships in purine biosynthesis of S. pombe (Fluri, Coddington & Flury, 1976), we found that ade mutants blocked directly after AIR were dark red, while those blocked before AIR remained white on this medium. All other strains tested, including wild type, showed a light red pigmentation. Pigmentation reactions for min mutants were the same as those observed for the corresponding allelic ade mutants.

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A. STRAUSS mutants mapping at ade loci in fact represent leaky ade mutants showing their auxotrophic character only under methionine supply. Wild-type like growth on MMA of these min mutants could be explained by a mutant enzyme activity which is reduced but still sufficient to support apparently full growth. Apart from this divergent growth behaviour on MMA, these min mutants resemble leaky ade mutants in every respect tested, i.e. in their adenine requirement under high methionine supply, in their chromosomal location and in their pigmentation. We assume that min alleles exist at all ade loci but that because of the limited number of mutants isolated, min mutants were discovered at only seven out of ten possible ade loci. The alleviating effect of adenine on the methionine sensitivity of other min mutants which do not map at ade loci remains unexplained by this hypothesis. The increase in the adenine requirement caused by methionine supply in min mutants and leaky ade mutants indicates that methionine or a metabolite derived from it inhibits adenine biosynthesis in S. pombe. Increase in pigment formation due to high methionine doses in all tested strains which are not blocked before AIR in adenine biosynthesis is consistent with this hypothesis. It further shows that the postulated inhibition of adenine biosynthesis takes place after the intermediate AIR, which is known to be accumulated and converted into the red pigment in ade6 and ade7 mutants (Strauss, 1972).
Although the physiological basis of the inhibition of adenine biosynthesis by methionine or its derivative is not obvious, it is likely that it is related to the biosynthesis of S-adenosylmethionine. This compound accumulates in yeast cells which are cultivated in medium containing methionine (Schlenk & DePalma, 1957). Enzymatic analysis will be required for confirmation and further characterization of the postulated inhibition.
Adenine biosynthesis appears also to be inhibited by methionine in Saccharomyces cerevisiae, as wild-type cells also form a red pigment when grown at high methionine concentrations and at low biotin concentrations (Cutts & Rainbow, 1950). Furthermore, the methionine-sensitive mutant met-i 3 behaves like the mini mutants of Schizosaccharomyces pombe in all ways tested (Meuris, 1969). This applies to the pigmentation under conditions of adenine limitation and to the reversion of methionine sensitivity by adenine as well as to the allelism with the structural gene for AIR-carboxylase (EC 4.1.1.21). The enzyme AIR-carboxylase in S. cerevisiae was shown to be coded by the ade2 locus (Yanulaitis, Nikolaeva & Smirnov, 1975) whilst indirect data suggest that in S. pombe it is coded by the ade6 locus (Strauss, 1972). In analogy to the situation for the mini mutants of S. pombe, the met-i 3 mutant of S. cerevisiae could thus be a leaky ade2 mutant which expresses its auxotrophic character only at high methionine supply.
The physiological basis for methionine sensitivity of min mutants of 8. pombe not mapping at ode loci is unknown. Some of these mutants are perhaps defective in methionine catabolism, as it was shown for one methionine-sensitive mutant of A. nidulans (Pieniazek et al. 1973).