GENETIC-CONTROL OF PURINE BIOSYNTHESIS IN YEAST PICHIA-METHANOLICA - IDENTIFICATION OF BIFUNCTIONAL GENE ADE7,4 CONTROLLING ACTIVITY OF GARAND AIR SYNTHETASES

A collection of induced and spontaneous mutants ade4, ade7, and ade4 a de7 of yeast Pichia methanolica has been investigated. By means of bio chemical and genetic methods, the ADE7,4 locus has been shown to be a complex structural gene controlling the second (E2) and the fifth (E5) steps of de novo purine biosynthesis. The ADE4 domain was shown to de termine phosphoribosyl aminoimidasol synthetase, or AIRS (E5), and the ADE7 domain-phosphoribosyl glycinamide synthetase, or GARS (E2). Gene tic properties of this locus, i.e., the control of two enzymatic activ ities, the presence of two domains, and the ability of interallelic co mplementation (IAC) in the mutants for the ADE4 domain determining the AIR synthetase activity, were analogous to the properties of homologi cal genes (ADE5/7 and ade1) in the Saccharomyces cerevisiae and taxono mically remote species Schizosaccharomyces pombe. This identification became the basis for cloning the ADE7,4 pm sequence from the genetic l ibrary of P. methanolica constructed in the YEp13 vector. The cloning was carried out in the S. cerevisiae recipient by complementation of t he ade7 mutation. Correspondence of the gene cloned was confirmed by a transformation of the ade4, ade7, and ade7,4 P. methanolica mutants.