Abstract
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.
Highlights
We first tried to isolate the human thymidylate synthase gene with a view to eventually isolating the mouse gene, since in practice cloning of the human gene is easier than thaotf the mouse gene, for the reasons mentioned below, and once the human gene has been cloned, it should not bedifficult to clone the mouse counterpart witha possible DNA sequence homologous to thatof the humangene
This paper describes the isolation of human thymidylate synthase cDNA clones from the human cDNA library using a DNAprobe, which was derived from one of the cloned thymidylate synthase gene fragments
The DNA was transferred to nitrocellulose filter and the resulting blots were hybridized with a nick-translated DNA fragment prepared from a cloned cDNA plasmid as described above
Summary
Dai AyusawaS, KeiichiTakeishi$, SumikoKanedaS, Kimiko ShimizuS, Hideki Koyamapll, and Takeshi SenoS. From onesuchtransformed cell clone, cloned recombinant X phages containing DNA fragments were obtained recently that were concluded by circumstantialgenetic evidence to have been derived from the human thymidylate synthase gene The cDNAlibrary has been prepared so as to contain preferably a full-length cDNA sequence and toallow expression of the inserted cDNA sequences in mammalian cells [11].The SV40-pBR322 shuttle vector usedin constructing the library contains a SV40 early region promoter and intron 5’ to thecDNA segment anda polyadenylation signal 3‘ to the cDNA segment These segments permit us to test for the function of the putative thymidylate synthasecDNA sequencedirectly in transformation assays and to identify and confirma full-length of the genomic sequence. This paper describes the isolation of human thymidylate synthase cDNA clones from the human cDNA library using a DNAprobe, which was derived from one of the cloned thymidylate synthase gene fragments
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