Cloning and expression of the OMP decarboxylase gene URA4 from Schizosaccharomyces pombe

Abstract

URA4, the gene coding for orotidine monophosphate decarboxylase (OMPdecase), has been cloned from the fission yeast by homologous complementation and restricted in an Escherichia coli-Schizosaccharomyces pombe (E. coli-S. pombe) replicative plasmid to a 1.76 kb HindIII fragment. This plasmid is maintained at a high copy number in S. pombe and allows OMPdecase expression in Saccharomyces cerevisiae (S. cerevisiae) as well as in E. coli. After characterisation by restriction mapping and Southern hybridisation, the cloned gene was used as a probe to measure URA4 transcription and to examine its regulation. Messenger RNA levels were measured by DNA/RNA filter-hybridisation with pulse labelled RNAs during 6-azauridine (6-AUR) inhibited growth in wild type and 6-AUR sensitive strains. We found that in S. pombe the OMP analogue 6-AUR does not regulate the level of OMPdecase formation as it does in S. cerevisiae but rather modifies the ratio of total polyA+ to polyA- RNAs in the cell. Based on these results and on corresponding enzyme activities this study demonstrates divergent pyrimidine pathway regulation in the two yeasts S. cerevisiae and S. pombe. Finally, we propose the use of the URA4 gene as a convenient selective marker for genetic engineering in S. pombe.