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pubmed:abstractText |
Resistance to copper's toxicity in yeast is controlled by the CUP1r locus. This gene was cloned by transforming sensitive recipients (cup1(8)) with a collection of hybrid DNA molecules, consisting of random yeast DNA fragments inserted into the vector YRp7. Four resistant transformants were studied in detail. Autonomously replicating or integrated by homologous recombination into chromosomal sites, the corresponding plasmids and several subclones confer resistance on sensitive recipients carrying the natural variant allele, cup1(8). Tetrad analysis and genetic mapping established that integration occurs typically at the cup1(8) site located 28 centimorgans distal to thr1, a chromosome VIII marker. Restriction endonuclease cleavage and electrophoretic mobility studies revealed that the CUP1r locus consists of a tandem array of repetitive units. Each unit is 1.95 kilobases in length and contains single sites for Kpn I and Xba I and two Sau3A sites. The sensitive allele represents one repeat and the resistant allele embraces 15 tandemly arrayed repeat units. Progressive selections in higher copper concentrations establish strains with markedly enhanced resistance. Resistance, we propose, is mediated by a gene amplification mechanism based on unequal sister chromatid exchange.
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