Unusual aspects of human thymidylate synthase in mouse cells introduced by DNA-mediated gene transfer.
Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme. In two transformants examined, however, neither was the dTTP pool elevated nor the dCTP pool decreased. DNA synthesis in permeabilized cells of a transformant was more efficient than that in the wild type with dATP, dGTP, dCTP, and dUMP as substrates, but this was not so when dUMP was replaced by dTTP. Unlike the mouse enzyme, the human enzyme in the transformants did not co-sediment with DNA polymerase alpha and thymidine kinase in a sucrose gradient, suggesting that the human enzyme is not incorporated into a multienzyme complex for DNA replication. The high levels of the human enzyme in the transformants were suppressed to various degrees by fusion with a wild type mouse line. No active hybrid dimer enzyme was found between the human and mouse enzymes, which each consist of two identical subunits. Thus, the human enzyme in the transformants seems to behave differently from the mouse enzyme and its overproduction seems to be necessary for supporting the normal growth of the transformants.
D000818:Animals; D002051:Burkitt Lymphoma; D002460:Cell Line; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism*; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology; D008780:Methyltransferases / Q000235:genetics*; D051379:Mice; D009154:Mutation; D009693:Nucleic Acid Hybridization; D013940:Thymidylate Synthase / Q000235:genetics*
D004273:DNA, Neoplasm; D004274:DNA, Recombinant; D008780:Methyltransferases; D013940:Thymidylate Synthase