CLONING AND MOLECULAR CHARACTERIZATION OF 3 GENES, INCLUDING 2 GENES ENCODING SERINE HYDROXYMETHYLTRANSFERASES, WHOSE INACTIVATION IS REQUIRED TO RENDER YEAST AUXOTROPHIC FOR GLYCINE

The genes encoding both the cytosolic and mitochondrial serine hydroxy methyltransferases (SHM2 and SHM1, respectively) and a third unidentif ied gene of the yeast Saccharomyces cerevisiae have been isolated and their nucleotide sequences determined. Analysis of the predicted amino acid sequence of the amino-terminal regions, sequence comparison with other genes encoding SHMT enzymes, and subcellular fractionation stud ies all suggested that the SHM1 gene encodes the mitochondrial SHMT, w hile the SHM2 gene encodes the cytosolic enzyme. The SHM2 gene but not the SHM1 gene has putative GCN4 sites upstream of the putative TATA b ox, suggesting regulation of its transcription by the general amino ac id control system. Yeast mutants with disruptions at each SHM gene and in both genes were constructed and all mutants had the same growth re quirements as the parental strains. Mutagenesis of the double-disrupte d, shm1 shm2 yeast yielded strains of a single complementation group t hat are auxotrophic for glycine. Complementation of the glycine auxotr ophy using a yeast genomic library retrieved the SHM1 and SHM2 genes a nd a third gene designated GLY1. Gene disruption studies demonstrated that inactivation of SHM1, SHM2, and GLY1 is required to yield yeast t hat are completely auxotrophic for glycine.