Isolation and Genetical Classification of Aromatic Amino Acid Auxotrophic Mutants in Schizosaccharomyces pombe

Abstract

173Mutants of Schizosaccharomyces pombe with single and multiple requirements for aromatic amino acids were isolated and allocated to 15 loci. Difficulties in cultivation of many of these auxotrophs were largely overcome by using a minimal medium with L-glutamic acid as sole nitrogen source. The locus-typical growth factor requirements of mutants allowed a preliminary allocation of genes to reaction sequences in aromatic amino acid biosynthesis. Several explanations for the occurrence of mutants with locus-atypical growth factor requirements are discussed. I N T R O D U C T l O NWe have attempted to isolate mutants of Schizosaccharomyces pombe auxotrophic for aromatic amino acids and aromatic vitamins. Mutants blocked in enzymes of the tryptophan-synthesizing branch of the pathway (Schweingruber & Dietrich, 1973) and others defective in isoenzymes controlling the first step of aromatic amino acid biosynthesis (Schweingruber & Wyssling, 1974) have already been isolated and characterized biochemically, but no mutants with multiple requirements for aromatic amino acids, as have been described in Neurospora crassa (Giles et al., 19671, Saccharomyces cerevisiae (De Leeuw, 1968) and Aspergillus nidulans (Roberts, 1969), have been isolated. Here we report on the isolation and genetical classification of such 5. pombe mutants as well as of others with single requirements for phenylalanine, tyrosine, tryptophan or p-aminobenzoic acid. METHODS Strains.The following strains of Schizosaccharomyces pombe stem from the silica gel stock collection of Professor U. Leupold, Institute of General Microbiology, University of Bern, Switzerland : wild-type strains 972 h-and 975 h+; tryptophan auxotrophic strains, originally isolated by Dietrich (1970), trpIA-BI h-/h+, trpIB-4 h-/h+, trpIC-3 h-/h+, trp2-2 h-/h+, trp3-23 h-/h+ and trp4-47 h-/h+.Media. Liquid yeast extract medium (YEL), malt extract agar (MEA) and minimal agar (MMA) were as described by Gutz et al. (1974). A slightly modified minimal medium (MMAg) was found to be more suitable for cultivation of aromatic amino acid auxotrophic mutants: in this, L-glutamic acid (10 g 1-1) replaced ammonium sulphate, Difco agar was used and the pH was adjusted to 5.5. Supplemented media (designated ' + aro') contained L-phenylalanine, L-tyrosine and L-tryptophan (all at M) and p-aminobenzoic acid M), added to the medium after autoclaving. Plates with supplemented media were stored at 4 "C and were used within 2 weeks.

Growth and the Environment of Schizosaccharomyces pombe

“…Growth on EMMG therefore has the advantage that both transporters can be used for uptake of basic amino acids. Empirical observations of a set of aromatic auxotrophic mutants show that EMMG supports much better growth than EMM2 in these mutants as well (Strauss 1979). For most researchers, however, EMMG is used because ura4-d18 mutants are fitter when grown with EMMG than with EMM2, presumably because uracil uptake exploits the same channels.…”

Canavanine Resistance and the Mechanism of Arginine Uptake in the Fission Yeast Schizosaccharomyces pombe

“…The use of glutamate has been reported (Strauss, 1979) to allow a higher rate of uptake of certain substrates, and this was presumably responsible for the increased sensitivity to canavanine. The wild-type 972 was completely inhibited at the lowest canavanine concentration tested during growth on glutamate.…”