Isolation of functional cDNA clones for human thymidylate synthase.
Thymidine auxotrophic mutants of mouse FM3A cells due to thymidylate synthase deficiency can be transformed into prototrophs by DNA-mediated gene transfer using total human DNA (Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., and Seno, T. (1983) J. Biol. Chem. 258, 48-53). From one such transformed cell clone, cloned recombinant lambda phages containing DNA fragments were obtained recently that were concluded by circumstantial genetic evidence to have been derived from the human thymidylate synthase gene (Takeishi, K., Ayusawa, D., Kaneda, S., Shimizu, K., and Seno, T. (1984) J. Biochem. (Tokyo) 95, 1477-1483). Using a DNA segment derived from the cloned genomic DNA fragment and free of repetitive sequences as a probe, functional cDNA corresponding to thymidylate synthase mRNA could be cloned from a cDNA library of SV40 transformed human fibroblasts constructed by Okayama and Berg (Okayama, H. and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289). The cloned cDNA plasmid containing an insert of approximately 1.7-kilobase transformed mouse thymidine auxotrophic mutant cells to thymidine prototrophic cells at a frequency of 2-3 transformants/micrograms of DNA/10(5) cells, a value almost comparable to the highest so far reported. The resultant transformants retained the introduced cDNA and expressed human thymidylate synthase protein sufficient for supporting normal growth of otherwise auxotrophic mouse cells.
D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D008970:Molecular Weight; D009154:Mutation; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification
D004247:DNA; D008780:Methyltransferases; D013940:Thymidylate Synthase; D004262:DNA Restriction Enzymes