Elevated dihydrofolate reductase messenger RNA levels in methotrexate-resistant BHK cells

Abstract

Polysomal RNA from cultured sublines of baby hamster kidney (BHK) cells directed protein synthesis in an in vitro system derived from wheat germ extract. One product of the in vitro synthesis was dihydrofolate reductase (DHFR), as confirmed by methotrexate-substituted Sepharose affinity chromatography followed by SDS-polyacrylamide slab gel electrophoresis and autoradiography of the proteins labeled with 35S-methionine. The DHFR synthesized in vitro comigrates in the gel with authentic BHK DHFR, indicating that the molecular weights and structures of the in vivo and in vitro enzymes are probably the same. Polysomal RNA obtained from the methotrexate-resistant BHK subline (A5), which possesses some 140 times higher DHFR levels than the methotrexate-sensitive parents subline (B1), directed the synthesis of approximately 70 times more DHFR per unit of total in vitro synthesized protein than did B1 polysomal RNA. Assuming then that the rates of translation of A5 and B1 DHFR mRNAs in the wheat germ cell-free system are the same, our results show that a major part of the high DHFR levels observed in A5 cells is due to the presence of elevated quantities of DHFR mRNA.