Conditional thymidine auxotrophic mutants of mouse FM3A cells due to thermosensitive thymidylate synthase and their prototrophic revertants.
A number of temperature-sensitive conditional thymidine auxotrophs were isolated from mutagenized mouse FM3A cells. Upon temperature shift from 33.5 degrees C to 39.5 degrees C, the mutant cells rapidly lost thymidylate synthase activity with concomitant decrease in intracellular dTTP and changes in other dNTP pools. Thymidylate synthase obtained from these mutants was inactivated in a manner following first order kinetics by heat treatment, which did not affect the parental enzyme. When bound covalently to 5-fluoro-2'-deoxyuridine 5'-[32P]monophosphate and 5,10-methylenetetrahydrofolate, the enzyme of one mutant migrated slower than the parental enzyme on nondenaturing polyacrylamide gel electrophoresis, whereas it did not do so on sodium dodecyl sulfate gel electrophoresis. Spontaneous prototrophic revertants were isolated from most of the mutant lines. In some revertants, the enzyme regained the heat resistance of the wild type completely or partially. In other revertants, the enzyme was overproduced, but its heat-sensitive nature was unaltered. The results demonstrate unequivocally that the conditional thymidine auxotrophy in the mutants was caused by thermosensitive thymidylate synthase due, at least in one particular line, to a missense mutation in its structural gene.
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D005816:Genetic Complementation Test; D006822:Hybrid Cells / Q000201:enzymology; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D013696:Temperature; D013936:Thymidine / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000378:metabolism*
D008780:Methyltransferases; D013940:Thymidylate Synthase; D013936:Thymidine