Overproduction Of Reductase Research Articles

Studies of the overproduction of dihydrofolate reductase by variant hamster cells in culture

Recent studies in this laboratory have concerned the regulation of dihydrofolate reductase in cultured hamster cells. Variant sublines highly resistant to Methotrexate due to markedly increased reductase activity were isolated. The reductase, purified by affinity chromatography, exists in hamster cells in two basic forms, which have different physical and kinetic properties but the same fingerprint patterns, and which cannot be interconverted. In the variant cells the main form has the same fingerprint pattern, and the minor form also is probably not altered. Turnover studies, first with antiserum to the enzyme and subsequently with affinity chromatography, indicate that the increase in reductase activity is entirely due to an increased rate of enzyme synthesis rather than decreased degradation. Polysomal RNA from the resistant cells, combined with a cell-free protein-synthesizing system, stimulates the in vitro production of reductase at an increased rate which accounts for half or more of the increased rate of enzyme production in the resistant cells. Possible mechanisms causing overproduction of the reductase are discussed, in addition to our current related studies.

HYBRIDIZATION OF HAMSTER CELLS WITH HIGH AND LOW FOLATE REDUCTASE ACTIVITY

These studies concern the question whether diffusible repressors control the syntheses of enzymes in mammalian cells. First, clonal sublines of baby hamster kidney (BHK 21/13) cells in culture were selected in stepwise fashion for resistance to aminopterin. These sublines survived in concentrations of aminopterin that were up to 10(4) times higher than those tolerated by wild-type cells because they contained up to 125 times as much folate reductase, probably due to overproduction of the enzyme. When five resistant sublines were hybridized to wild-type lines, 32 of 35 hybrid clones contained intermediate levels of reductase activity. This suggests that overproduction of reductase is not due to loss of a diffusible repressor.