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-IIa locus, exhibits selective and tandem amplification in many wild type strains of C. glabrata. The present paper demonstrates that the amplified MT-IIa 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-IIa gene were localized to a 457-bp segment downstream from the MT-IIa 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-IIa gene is used. Transformation of C. glabrata with plasmids carrying the 2 microns circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2 microns plasmids replicate only to a limited extent in C. glabrata. Conversely, a C. glabrata plasmid carrying three copies of the MT-IIa gene was able to transform S. cerevisiae.
Mehra
R K
RK
Department of Medicine, University of Utah Medical Center, Salt Lake City 84132.
Thorvaldsen
J L
JL
Macreadie
I G
IG
Winge
D R
DR
D001483:Base Sequence; D002175:Candida / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004261:DNA Replication / Q000235:genetics*; D005784:Gene Amplification; D005800:Genes, Fungal; D008668:Metallothionein / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014170:Transformation, Genetic
D009838:Oligodeoxyribonucleotides; D008668:Metallothionein