MULTICOPY CUP1 PLASMIDS ENHANCE CADMIUM AND COPPER RESISTANCE LEVELS IN YEAST

A 3.3 kb fragment of yeast genomic DNA was isolated by screening a genomic library constructed in the high copy number 2 micron plasmid YEp351 vector for clones capable of enhancing the degree of resistance of Saccharomyces cerevisiae strain MW3070-8B to cadmium. The insert contained two complete copies of the CUP1 gene open reading frame (183 bp), including the upstream promoter sequences (450 bp) with two conserved metal responsive cis-acting elements. Northern analysis showed that addition of cadmium (0.02-mu-M) or copper (50-mu-M) to overnight liquid cultures of yeast induced expression of CUP1 transcripts from both chromosomal and plasmid-borne gene copies. The cloned 3.3 kb DNA in a high copy number plasmid restored copper resistance to the sensitive strain LS70-3B-DELTA, deleted for the CUP1 gene (cup 1-DELTA), but failed to restore cadmium resistance. Thus, CUP1 gene expression in yeast appears to be influenced differently by cadmium and copper ions. Resistance to heavy metal poisoning resulted from enhanced gene product levels attributable to amplification of the CUP 1 gene as well as to increased transcriptions. Two distinct gene product levels mediate cadmium and copper resistance; a higher gene product level was required to confer cadmium resistance.