The possible role of metallothioneins in copper tolerance of Silene cucubalus

The subcellular distribution and chemical form of Cd in bean plants grown in nutrient solutions containing Cd were investigated. Cd was accumulated mainly in roots and to a minor extent in leaves. Subcellular fractionation of Cd-containing tissues (pH 7.5) showed that more than 70% of the element was localized in the cytoplasmic fraction in roots as well as in leaves. Little Cd (8 to 14%) was bound either to the cell wall fraction or to the organelles. Gel filtration of the soluble fraction showed Cd to be associated mainly with 5,000 to 10,000 molecular weight components in roots, and 700 to 5,000 molecular weight components in leaves. Small amounts of Cd were found in the high molecular weight proteins (molecular weight 150,000). Only traces of Cd could be detected as a free ion. Chemical characterization of the low molecular weight components resulted in the identification of nine amino acids which were identical in roots and leaves. Cd in bean plants is assumed to be bound to peptides and/or proteins of low molecular weight.

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.