Cloning system for Candida glabrata using elements from the metallothionein-II a-encoding gene that confer autonomous replication

Abstract

The yeast Candida glabrata harbors two distinct families that encode metallothioneins (MTs). One of these loci, the MT-II a locus, exhibits selective and tandem amplication 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 2μ circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2μ 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.