CLONING, AND MOLECULAR CHARACTERIZATION OF THE GCV1 GENE ENCODING THEGLYCINE CLEAVAGE T-PROTEIN FROM SACCHAROMYCES-CEREVISIAE

We have isolated the gene encoding the glycine cleavage T-protein (GCV 1) of the yeast Saccharomyces cerevisiae and shown through gene disrup tion and enzyme assays that inactivation of GCV1 destroys glycine clea vage function. A DNA fragment encoding the GCV1 gene was cloned by PCR amplification using degenerate oligodeoxyribonucleotides, and the clo ned fragment was used as a probe to isolate the complete gene from a y east genomic library. Growth with glycine stimulated expression of the GCV1 gene as determined by Northern analysis and increased the beta-g alactosidase activity of a GCV1-lacZ fusion 30-fold. The URA3 gene was inserted into the coding sequence of GCV1 and the resulting construct was used to disrupt the chromosomal GCV1 gene in a diploid strain of yeast. gcv1::URA3 haploid derivatives grew normally or only slightly m ore slowly than the isogenic wild-type haploids. All gcv1 strains stud ied were unable to grow on glycine as a sole nitrogen source and lacke d glycine cleavage enzyme activity. Growth of shm1 shm2 mutants was st imulated by glycine, whereas glycine could not supplement the growth o f the isogenic gcv1 strain.