A method is described for the assay of thymidylate synthetase activity in crude ex tracts of Ehrlich ascites tumor. The method involves the use of tritiated deoxyuridine5'-phosphate as substrate. The inhibition of thymidylate synthetase by 5-fluorodeoxyuridine-5'-phosphate was measured in extracts from one 5-fluorouracil-sensitive and four drug-resistant lines of Ehrlich ascites tumor. A high d...

The 1017-bp intron within the cloned phage T4 td gene was deleted by oligonucleotide-directed mutagenesis. Induction of thymidylate synthase activity and mature td mRNA from this intronless construct (pKTd delta I) was compared both in vivo and in vitro with expression from plasmids bearing td genes in which the introns contain either no change (pKTd2), an XbaI linker inserted about 200 nucleot...

BACKGROUND Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. RESULTS Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahy...

When 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine is administered to Drosophila larvae together with 5-fluorouracil, many of the adult flies show growth lesions in the form of extra bristles and other supernumerary wing structures. The hypothesis that the growth lesions are the result of thymidine analog incorporation in DNA and that the effects of this incorporation are increased by feedin...

We have developed a simple procedure to identify protein homologs in genomic databases. The program, called ORF, is based on comparisons of predicted secondary structure. Protein structure is far better conserved than amino acid sequence, and structure-based methods have been effective in exploiting this fact to find homologs, even among proteins with scant sequence identity. ORF is a secondary...

Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequ...

Objective: To analyze the impact of mRNA expression of oral fluoropyrimidine (S-1) metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase [OPRT]), on treatment outcomes in locally advanced gastric cancer patients receiving preoperative S-1 combined with oxaliplatin chemotherapy. Methods: Preoperative stage III gastric ca...

Trypanosomatid protozoans depend upon exogenous sources of pteridines (pterins or folates) for growth. A broad spectrum pteridine reductase (PTR1) was recently identified in Leishmania major, whose sequence places it in the short chain alcohol dehydrogenase protein family although its enzymatic activities resemble dihydrofolate reductases. The properties of PTR1 suggested a role in essential pt...

Taking trimethoprim as the selective agent in the presence of thymine, we adapted to Bacillus subtilis a selection procedure depending on the peculiar organisation of the one-carbon metabolism. The corresponding pathways couple synthesis of thymine to tetrahydrofolate consumption as a substrate of the reaction mediated by thymidylate synthase, instead of being a co-enzyme as in the other reacti...

The combination of fluoropyrimidines and radiation has resulted in increased control of colorectal cancer in the clinic, but the basic mechanism of the interaction is not understood clearly. Preliminary work in our laboratory showed that 2-h exposures of HT 29 human colon carcinoma cells to relatively low levels of 5-fluorodeoxyuridine resulted in extended thymidylate synthase inhibition after ...