Cloning, and molecular characterization of the GCV1 gene encoding the glycine cleavage T-protein from Saccharomyces cerevisiae.
We have isolated the gene encoding the glycine cleavage T-protein (GCV1) of the yeast Saccharomyces cerevisiae and shown through gene disruption and enzyme assays that inactivation of GCV1 destroys glycine cleavage function. A DNA fragment encoding the GCV1 gene was cloned by PCR amplification using degenerate oligodeoxyribonucleotides, and the cloned fragment was used as a probe to isolate the complete gene from a yeast genomic library. Growth with glycine stimulated expression of the GCV1 gene as determined by Northern analysis and increased the beta-galactosidase 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 more slowly than the isogenic wild-type haploids. All gcv1 strains studied were unable to grow on glycine as a sole nitrogen source and lacked glycine cleavage enzyme activity. Growth of shm1 shm2 mutants was stimulated by glycine, whereas glycine could not supplement the growth of the isogenic gcv1 strain.
McNeil
J B
JB
Department of Medical Genetics and Microbiology, University of Toronto, Ont. Canada.
Zhang
F
F
Taylor
B V
BV
Sinclair
D A
DA
Pearlman
R E
RE
Bognar
A L
AL
D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D050960:Aminomethyltransferase; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D005656:Fungal Proteins / Q000235:genetics*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D012333:RNA, Messenger / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D002352:Carrier Proteins; D003470:Culture Media; D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C064525:glycine cleavage system; D000594:Amino Acid Oxidoreductases; D014166:Transferases; D050960:Aminomethyltransferase; C104785:GCV1 protein, S cerevisiae