Isolation of the yeast phosphoglyceromutase gene and construction of deletion mutants

Abstract

The PGM1 gene (also called GPM; Fraenkel 1982) coding for phosphoglyceromutase was isolated by functional complementation. When present on a multicopy vector and introduced into yeast cells it led to an about eightfold increase in specific enzymatic activity. This apparent overproduction was confirmed by SDS-polyacrylamide gel electrophoresis of crude extracts and at the transcriptional level by Northern analysis. By subcloning of the yeast DNA insertions of the plasmids originally isolated the PGM1 coding region was located within a 1.3 kb SalI-HindIII fragment. Integration at the chromosomal locus confirmed that the PGM1 gene had indeed been isolated. Southern analysis of genomic digests showed the same restriction patterns as the cloned sequences. However, a BamHI restriction polymorphism was observed. Furthermore, a repetitive element was found in the PGM1 flanking region. Finally, the chromosomal copy of the gene was deleted by replacement with a URA3 marker. The deletion mutants showed that the gene is not essential for yeast growing in the presence of a combination of glycerol and ethanol. However, growth was inhibited by glucose and neither glycerol nor ethanol alone were sufficient to support growth.