THYMIDYLATE SYNTHASE GENE AMPLIFICATION IN FLUORODEOXYURIDINE-RESISTANT MOUSE-CELL LINES

Abstract

Fluorodeoxyuridine-resistant mouse fibroblast (LU3-7) and neuroblastoma (FUdR-R) cell lines were isolated that overproduce thymidylate synthase and the mRNA for this enzyme up to 50-fold as compared to the parental cell lines. cDNA corresponding to mouse thymidylate synthase mRNA c[complementary]DNA was cloned as a hybridization probe in Southern blot analysis of DNA from the parental and overproducing cell lines. These analyses showed that the thymidylate synthase gene is amplified 50- to 100-fold in the LU3-7 cells and about 30-fold in FUdR-R cells when compared to the respective parental cells. The sizes of the restriction fragments were the same in the parental and overproducing cells of each type, suggesting that extensive rearrangements did not occur in the vicinity of the thymidylate synthase gene during the amplification process. Not all of the fragments in the parental cells were amplified in the overproducing cells, suggesting that there may be multiple genes or pseudogenes for the enzyme. Restriction fragment length polymorphisms were detected when analyzing DNA from several different mouse cell lines. When LU3-7 cells were grown in the absence of selective pressure, the level of thymidyate synthase overproduction and the number of copies of the thymidylate synthase gene decreased in parallel.