Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.With the exception of (deoxy)thymidylate kinase (dTMP kinase or TMK), which catalyzes the phosphorylation of dTMP to form dTDP in the dTTP synthesis pathway, all of the Escherichia coli enzymes of the metabolic pathways for the de novo synthesis of deoxynucleotide precursors of DNA have been well characterized genetically and biochemically (34). dATP, dCTP, and dGTP are derived from the corresponding deoxyribonucleoside diphosphates by phosphorylation catalyzed by the nonspecific nucleoside diphosphate kinase. Because thymine deoxyribonucleotides have no ribonucleotide counterpart, additional reactions are required for dTTP synthesis involving successively dCTP deaminase, dUTPase, thymidylate synthase, dTMP kinase, and nucleoside diphosphate kinase to convert dCTP to dTTP. Since conversion of dTDP to dTTP is catalyzed by the nonspecific nucleoside diphosphate kinase, the dTMP kinase is the last specific enzyme of both de novo and salvage pathways of dTTP synthesis. Because the overall control of DNA synthesis is regulated in part by the finely adjusted pool of dTTP, it would be crucial to explore the expression and the regulation of the E. coli dTMP kinase gene.For Saccharomyces cerevisiae, knowledge of the function of the dTMP kinase gene (cdc8) comes from the studies of a cdc8 temperature-sensitive cell cycle mutant. The transcription of the yeast dTMP kinase gene has been shown to be cell cycle regulated (peaking at the S phase) and coexpressed with DNA ligase and thymidylate sy...

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.