Isolation of the Cryptococcus humicolus URA3 gene encoding orotidine-5′-phosphate decarboxylase and its use as a selective marker for transformation

Abstract

A transformation system for a yeast, Cryptococcus humicolus, was constructed. As a selectable marker, the URA3 gene encoding orotidine-5′-phosphate decarboxylase (OMPdecase) was isolated from a C. humicolus genomic DNA library, and the equivalent cDNA was cloned. The coding region encompasses a polypeptide of 269 amino acids interrupted by two introns, which were located at the same positions as observed in the equivalent genes of some filamentous fungi. The deduced amino acid sequence showed significant homology to those of OMPdecases from other fungal species. Although no canonical TATA and CHAT sequences and polyadenylation sequence are in the flanking regions, two C+T-rich sequences are observed in the 5′-flanking region. The cDNA of the URA3 gene of C. humicolus was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae. As a host, five uracil auxotrophic mutants were isolated by the selection of ethyl methanesulfonate mutagenized cells on 5-fluoroorotic acid. Three of them could be transformed to Ura + phenotype with a linearized URA3-harboring vector using electroporation, and the best transformation frequency was 14 transformants per μg of DNA. Southern blot analysis of five independent transformants showed the integration of the vector into the host chromosomal DNA at the URA3 locus in one transformant, and also the integration at ectopic sites and the modified extrachromosomal forms in others.