Selective Multiplication of Dihydrofolate Reductase Genes in Methotrexate-resistant Variants of Cultured Murine Cells*

The rate of dihydrofolate reductase synthesis in the AT3000 line of methotrexate-resistant murine Sarcoma 180 cells is approximately 200to 250-fold greater than that of the sensitive, parental line. We have purified cDNA sequences complementary to dihydrofolate reductase mRNA and subsequently used this probe to quantitate dihydrofolate reductase mRNA and gene copies in each of these lines. Analysis of the association kinetics of the purified cDNA with DNA from sensitive and resistant cells indicated that the dihydrofolate reductase gene is selectively multiplied approximately 200-fold in the resistant line. A similar analysis of a partially revertant line of resistant ceils indicated that the loss of resistance observed when the AT-3000 line is grown in the absence of methotrexate is associated with a corresponding decrease in the dihydrofolate reductase gene copy number. In each of these lines the relative number of dihydrofolate reductase gene copies is proportional to the cellular level of dihydrofolate reductase and dihydrofolate reductase mRNA sequences. We have also studied parental and methotrexate-resistant lines of L1210 murine lymphoma cells. Both resistance and an associated 35-fold increase in the level of dihydrofolate reductase appear to be stable properties of the resistant L1210 line since we find no decrease in either parameter in over 100 generations of growth in the absence of methotrexate. Once again, we find that the increased levels of dihydrofolate reductase in the methotrexate-resistant IJ210 line are associated with a proportional increase in the number of dihydrofolate reductase gene copies. In this case the dihydrofolate reductase gene copy number appears to be relatively stable in the resistant line. Therefore, we conclude that selective multiplication of the dihydrofolate reductase gene can account for the overproduction of dihydro-