CLONING SYSTEM FOR CANDIDA-GLABRATA USING ELEMENTS FROM THE METALLOTHIONEIN-IIA-ENCODING GENE THAT CONFER AUTONOMOUS REPLICATION

The yeast Candida glabrata harbors two distinct gene families that encode metallothioneins (MTs). One of these loci, the MT-II(a) locus, exhibits selective and tandem amplification in many wild type strains of C. glabrata. The present paper demonstrates that the amplified MT-II(a) gene contains autonomously replicating sequences (ARS). These ARS elements have been used to construct vectors capable of replicating in C. glabrata. The ARS element(s) in the MT-II(a) gene were localized to a 457-bp segment downstream from the MT-II(a) coding sequence. Although plasmids containing this fragment transform C. glabrata with high frequency, the stability of the transformants and the copy number of the plasmid improve when the entire 1.25-kb MT-II(a) gene is used. Transformation of C. glabrata with plasmids carrying the 2-mu-circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2-mu-plasmids replicate only to a limited extent in C. glabrata. Conversely, a C. glabrata plasmid carrying three copies of the MT-II(a) gene was able to transform S. cerevisiae.