Rapid spontaneous dihydrofolate reductase gene amplification shown by fluorescence-activated cell sorting

We have determined whether the gene encoding dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP⁺ oxidoreductase, EC 1.5.1.3) varies spontaneously in gene copy number in cells in vitro. Cells were stained under nonselective conditions with fluoresceinated methotrexate, which binds quantitatively to dihydrofolate reductase. Cells with the highest fluorescence were collected by a fluorescence-activated cell sorter and subsequently grown in the absence of methotrexate. At no time during the experiment were the cells placed under metabolic stress. After 10 successive rounds of growth and sorting, the derived population showed a 50-fold increase in fluorescence intensity, was highly resistant to methotrexate, and was amplified 40-fold in content of dihydrofolate reductase gene. We also found that cells already having amplified genes can undergo increases or decreases in their fluorescence and in gene copy number even more rapidly (at rates as high as 3 x 10^-2 amplification events per cell division) than do parental cells (ca. 10^-3 events per division). We therefore conclude that gene amplification can occur spontaneously in cells and that the rate of its occurrence varies with gene copy number.