A TRANSFORMATION SYSTEM FOR THE YEAST CANDIDA-UTILIS - USE OF A MODIFIED ENDOGENOUS RIBOSOMAL-PROTEIN GENE AS A DRUG-RESISTANT MARKER AND RIBOSOMAL DNA AS AN INTEGRATION TARGET FOR VECTOR DNA

We have developed a transformation system for the yeast Candida utilis , A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transforma tion are not available, A gene encoding the ribosomal protein L41 was cloned from C, utilis, which is sensitive to cycloheximide, and used a s a marker gene conferring cycloheximide resistance after modification of its amino acid sequence, The marker gene was constructed by substi tution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56t h amino acid residue of L41 is responsible for the cycloheximide sensi tivity of various organisms (S, Kawai, S, Murao, hi. Mochizuki, I, Shi buya, K, Yano, and M, Takagi, J, Bacteriol, 174:254-262 1992), The rib osomal DNA (i,e,, DNA coding for rRNA) of C. utilis was also cloned an d used as a multiple copy target for the integration of vector DNA int o the genome, which resulted in a high transformation efficiency, Tran sformants were obtained by electroporation with a maximum efficiency o f approximately 1,400 transformants per 1 mu g of linearized DNA carry ing the gene for cycloheximide resistance and part of the ribosomal DN A, No transformants were obtained with intact plasmids, Multiple copie s of the linearized plasmid were integrated into the host chromosome b y homologous recombination, Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that t here are two copies of gene for the L41 protein per cell, suggesting t hat C. utilis is diploid, Transformants were obtained from a variety o f C. utilis strains, indicating that this method is applicable to the transformation of other C, utilis strains, even though there is signif icant heterogeneity in chromosomal karyotypes among these strains.