Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.
ABSTRACT The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 184.108.40.206) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.
Article: Thymidylate synthetase.Advances in enzymology and related areas of molecular biology 02/1973; 38:235-92.
Article: Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event.Biochemistry 11/1970; 9(22):4428-40. · 3.42 Impact Factor
Article: Production of a functional eukaryotic enzyme in Escherichia coli: cloning and expression of the yeast structural gene for imidazole-glycerolphosphate dehydratase (his3).[show abstract] [hide abstract]
ABSTRACT: A cloned segment of yeast DNA containing the structural gene for imidazoleglycerolphosphate dehydratase (D-erythro-imidazoleglycerolphosphate hydro-lase, EC 220.127.116.11) is transcribed and translated in Escherichia coli with sufficient fidelity to produce functional enzyme. This segment of yeast DNA was isolated as a viable molecular hybrid of bacteriophage lambda (lambdagt-Sc2601) which complements a nonrevertible hisB auxotroph of E. coli lacking dehydratase activity. The equivalent segments of DNA cloned from two independent his3 mutants of yeast lacking IGP dehydratase activity do not complement the hisB auxotroph. The two nonfunctional his3 alleles cloned in bacteriophage lambda can be recombined in E. coli to generate a hybrid phage which complements the hisB auxotroph. The dehydratase activity produced in E. coli by the cloned segment of yeast DNA strongly resembles the activity found in yeast.Proceedings of the National Academy of Sciences 01/1978; 74(12):5255-9. · 9.68 Impact Factor