ISOLATION AND STUDY OF MUTANTS FROM MAMMALIAN CELLS IN CULTURE

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Abstract
The antibiotic puromycin (8 ug/ml) was added to the growth medium of heavy growth cultures of the susceptible parent fibroblast-like cells (AMK 2-2). When about 95% of the cells had been killed (about 24 hr), the medium was replaced with fresh puromycin-free growth medium and the culture grown to its original density. The process of growth and selection was repeated through 6 cycles until some of the cultures (Pmr-1) were resistant and capable of growth in the presence of 12 ug/ml of the antibiotic. Cells resistant to higher levels (Pmr-2) were selected from the less resistant mutant cultures with growth medium containing 40 [mu]g/ml. The Pmr-2 cells were obtained with ease after a single cycle of selection and regrowth. Examination of the descendants of the wild type cells cloned in the absence of puromycin revealed 2 major classes, a highly sensitive one and a less sensitive one. The isolation of 4 heritably different cultures with gradually increasing resistance to puromycin suggests that the origin of the most resistant cells involves sequential mutational events. In contrast, cells selected in the presence of 1 [mu]g/ml of 8-azoguanine grew in a medium containing a 100 times greater concentration of the analogue and indicated a "one-step" increase in resistance with no evidence for cells with intermediate levels of resistance.