Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) 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.
Mol Cell Biol 1982 Apr
PMID:Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae. 628 38

The 7.8-kilobase HindIII insert in phage lambda NM589thyA [Borck, K., Beggs, J.D., Brammar, W.J., Hopkins, A.S. & Murray, N. (1976) Mol. Gen. Genet. 146, 199] was confirmed as originating from Escherichia coli by hybridization analysis and was shown to encode the thymidylate synthetase (5,-10-methylenetetrahydrofolate:dUMP C-methyltransferase EC 2.1.1.45) of E. coli K-12 by using biochemical, structural, and immunologic criteria. The 7.8-kilobase insert was reduced in size to a quasi-random population of DNA subfragments by partial digestion with the 4-base-pair recognition enzymes Alu I and Hae III. A clone containing a 1.1- to 1.2-kilobase fragment that encompassed the gene was obtained from this mixture by selecting for Thy+ recombinants. Fusion of this DNA fragment to the phage lambda rho L promoter in plasmid pKC30 revealed the direction of transcription of the thyA gene, and, in a phage lambda lysogen containing a thermolabile repressor, intracellular synthetase levels were increased about 700-fold. The enzyme was purified to homogeneity from this source by affinity chromatography, and some of its properties are described.
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PMID:Characterization of the Escherichia coli thyA gene and its amplified thymidylate synthetase product. 634 Jan 11

T4-infected cells, plasmolysed 15 min after infection, incorporate low concentrations (less than 20 microM) of deoxythymidine (TdR) into DNA at a significantly greater rate than dTMP, dTTP or thymine. At higher concentrations (greater than 40 microM), dTMP incorporation rate is high, approaching that of TdR at 200 microM. TdR is selectively incorporated at all concentrations tested, and is not inhibited by the other thymine containing DNA precursors. Incorporation of low concentrations of TdR requires the T4-induced thymidine kinase (tk) and is not significantly affected by the presence or absence of T4-induced thymidylate synthetase (td). We show that, in T4-infected plasmolysed cells, exogenously added TdR is preferentially incorporated into short DNA fragments during short pulse times. To explain these and other data a model is proposed in which thymidine plays a modulatory role between leading and lagging strand precursor feeds.
Mol Gen Genet 1983
PMID:Incorporation of thymine-containing DNA precursors in wild-type and mutant T4-infected plasmolysed cells. 635 61

Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.
Mol Cell Biol 1984 Dec
PMID:Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae. 639 9

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.
Mol Cell Biol 1984 Dec
PMID:Thymidine-requiring mutants of Dictyostelium discoideum. 652 88

Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating substrate concentration to initial velocity was changed from a sigmoidal shape to a hyperbolic one by the activator dCTP, while the inhibitor TTP increased the sigmoidicity of the curve. The molecular weight of deoxycytidylate deaminase was unchanged in the presence of allosteric effectors, indicating that aggregation-disaggregation is not the basis of regulation. Deoxycytidylate deaminase exhibited the greatest affinity for the substrate dCMP, with lesser affinity for ara-CMP, and least affinity for CMP. Ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 4 microM. These data indicate that human neoplastic cell deoxycytidylate deaminase is a highly regulated allosteric enzyme, which is likely to have a significant influence on cellular dUMP, dCTP and TTP pools. These findings further suggest, that the enzyme through its influence on dUMP levels is likely to modulate the biochemical effects of pyrimidine antimetabolites active against the thymidylate synthetase reaction and in the presence of elevated dCTP pools will promote deamination of ara-CMP to the inactive ara-UMP.
Mol Cell Biochem 1983
PMID:Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells. 658 81

The purpose of this study was to compare the pools of free FdUMP derived from 5-fluorouracil (FUra) and of dUMP synthesized de novo in Hep-2 and S-180 cells, their relationship to inhibition of thymidylate synthase (dTMP synthase; EC 2.1.1.45), and the effect of excess folinic acid (CF) on these parameters. These cells differ 50-fold in their sensitivity to FUra and, in the absence of thymidine, dTMP synthase is the growth-limiting site of action of FUra in S-180 cells, but in Hep-2 cells this site becomes growth-limiting only in the presence of excess folates. In both cells after a 3-hr incubation with varied concentrations of FUra, FdUMP comprised only 0.1-0.2% of the total acid-soluble pools derived from FUra. The changes in dUMP and FdUMP pools paralleled each other, dUMP being 1000-2000 times higher than FdUMP. The pools of dUMP increased only when dTMP synthase was significantly inhibited. This occurred in S-180 cells above 3 microM FUra and in Hep-2 cells above 30 microM, where the residual dTMP synthase was similar in both cells. Under these conditions, the dUMP and FdUMP pools in Hep-2 cells were 2 and 4 times higher, respectively, than in S-180 cells. After FUra removal, both pools continued to increase, dUMP and FdUMP pools in Hep-2 cells rising 6-fold and 10-fold higher, respectively, than in S-180 cells. The dTMP synthase inhibition and the high nucleotide pools in Hep-2 were short-lived, whereas in S-180 cells the inhibition and the pools were maintained longer. Excess CF retarded the recovery of dTMP synthase after FUra removal only in Hep-2 cells and led to a further increase in dUMP and FdUMP pools in these cells, while having no effect in S-180 cells. These data indicate that a high capacity of cells to accumulate free FdUMP does not alone guarantee that dTMP synthase inhibition will be growth-limiting. The relationship shown here between excess CF, dTMP synthase recovery, and the nucleotide pools suggests that some cell types, such as Hep-2, in spite of high levels of FdUMP, require in addition an excess of folates to retard dTMP synthase recovery and make it growth-limiting.
Mol Pharmacol 1984 Mar
PMID:Relationship of dUMP and free FdUMP pools to inhibition of thymidylate synthase by 5-fluorouracil. 660 49

The binding of 5-fluoro-2'-deoxyuridylate, methylenetetrahydrofolate, and thymidylate synthetase in a ternary complex results in enzyme inhibition and is a major component of 5-fluorouracil cytotoxicity in some cells. The amount of 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase in several human gastrointestinal tumor cell lines following 5-fluorouracil exposure was determined, using Sephadex G-25 chromatography and high-pressure chromatographic analysis. These data were compared with previously determined values for thymidylate synthetase levels in control cultures not exposed to 5-fluorouracil. In HuTu 80 cells, the amount of 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase represented 16% of the total amount of enzyme present in untreated cells. The values for 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase in the WIDR and HT 29 cell lines (57 and 46 fmoles/10(5) cells, respectively), however, exceed by 3- to 6-fold the total amount of this enzyme found in untreated cells. The presence of increased levels of thymidylate synthetase protein in these cell lines was confirmed by sodium dodecyl sulfate gel electrophoresis. Measurements of thymidylate synthetase levels following exposure of cells to cycloheximide demonstrated that thymidylate synthetase complexed to 5-fluoro-2'-deoxyuridylate has increased stability as compared with uncomplexed enzyme. The level of thymidylate synthetase (bound and free) present in WIDR cells was measured following removal of FUra from the media. After 48 hr, the level of bound enzyme had fallen from 53 to 14 fmoles/10(5) cells, whereas free enzyme, which was undetectable after a 24-hr exposure to FUra, returned to 60% of its level in untreated cells.
Mol Pharmacol 1984 Jan
PMID:Increased levels of thymidylate synthetase in cells exposed to 5-fluorouracil. 670 32

Thymidylate synthetase and dihydrofolate reductase exist as a bifunctional protein in a number of species of protozoa which span diverse groups of the subkingdom. The enzymes copurify upon gel filtration and on affinity chromatography columns specific for dihydrofolate reductase. The bifunctional protein has been found in species of Crithidia, Leishmania, Trypanosoma, Plasmodium, Eimeria, Tetrahymena and Euglena. For reasons unknown, neither enzyme could be detected in Entamoeba histolytica or E. invadens. Since neither enzyme has yet been found as a separate protein in protozoa, it is likely that the bifunctional protein is widespread among these primitive eukaryotes. In most cases, the apparent size of the native protein is approximately twice that of the subunit possessing thymidylate synthetase. Further, with one exception, the subunit sizes are close to the sum of the subunit sizes of the separate enzymes found in other sources.
Mol Biochem Parasitol 1984 Apr
PMID:A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. 674 82

Unlike wild-type Saccharomyces cerevisiae, yeast cells carrying the tup 7 mutation are able to take up exogenously-supplied dTMP. The tup 7 mutant was also found to be dramatically sensitive to growth inhibition by FdUMP and BrdUMP. The exclusive mode of action of FdUMP in such strains was shown to be inhibition of thymidylate synthetase. Spontaneously-arising derivatives resistant to FdUMP and BrdUMP were isolated from the tup7 strain. Genetically, these mutations were recessive and defined three complementation groups (fdr1, fdr2, and bdr2), unlinked to the tup7 locus and to each other. No resistance mutations were obtained which mapped at the structural gene for thymidylate synthetase. Biochemical analysis of cells carrying these mutations showed that in the case of fdr2 and bdr2, in addition to an inability to transport dTMP, acid and alkaline phosphatase levels were affected, indicating that phosphatase expression and 5'-mononucleotide permeability are coordinately controlled. In contrast, the fdr1 mutation and a previously identified suppressor of dTMP-permeability, sot1, affected only 5'-mononucleotide uptake and may define components of the permease responsible for dTMP entry.
Mol Gen Genet 1982
PMID:Effect of halogenated pyrimidine 5'-mononucleotides on dTMP-permeable yeast strains and the isolation and characterization of resistant mutants. 675 56


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