Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The molecular karyotypes of five species of Leishmania were studied by pulsed field gradient gel electrophoresis (PFGGE) of chromosome-sized DNA bands. Each species exhibits a unique pattern of 22-28 bands in the size range approximately 200-2200 kb whereas strains of one species exhibit similar karyotypes. Analysis of the behaviour of kinetoplast DNA during PFGGE showed that minicircle DNA remains confined to the gel slot but a proportion of the maxicircle DNA fractionates as a low molecular weight band below band 1. The band location of genes for alpha and beta tubulin, the 5' spliced leader sequence (5'SL), heat shock proteins 70 (hsp 70) and 83 (hsp 83) and thymidylate synthase-dihydrofolate reductase (TS-DHFR) were analysed. Housekeeping genes are not clustered in Leishmania but are found on at least 7 bands in L. major. The hsp 83 gene is linked to the tandemly repeated beta tubulin allele on band 21 in L. major. Among different species, the location of the unlinked hsp 83 and hsp 70 genes is conserved whereas the TS-DHFR and 5'SL sequences are found on bands of varying size. The 5'SL gene may be rearranged in L. enriettii and two 5'SL loci were identified in L. donovani and L. tropica. The conservation of loci in strains of L. major suggests that the chromosomal genetic linkage map should be a reliable marker for identifying unknown isolates of Leishmania. Sequences on one band in L. mexicana sp. were shared among several bands and distributed on homologous and non-homologous bands in other species showing that DNA sequences are rearranged during speciation in Leishmania.
Mol Biochem Parasitol 1987 Oct
PMID:Molecular karyotype of five species of Leishmania and analysis of gene locations and chromosomal rearrangements. 282 21

We have analyzed the cell cycle effects that different domains of the adenovirus E1A proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells. A virus containing a mutation in this region was still able to stimulate DNA synthesis efficiently. A third domain, unique to the 13S protein, was required for the accelerated activation of the cellular thymidylate synthase gene in a manner similar to the 13S-dependent stimulation of adenovirus early region genes.
Mol Cell Biol 1987 Feb
PMID:Different functional domains of the adenovirus E1A gene are involved in regulation of host cell cycle products. 288 Nov 97

Cytotoxicity to 5-fluoro-2'-deoxyuridine (FdUrd) derives from its conversion to 5-fluorodeoxyuridine-5'-monophosphate, which binds to and inhibits thymidylate synthase (TS) in the presence of the cofactor, 5,10-methylenetetrahydrofolate. We have selected FdUrd-resistant variants of the human cell line HEp-2 following adaptation to stepwise increases in drug concentration. In the initial selection, maximal drug resistance was associated with a 26-fold increase in the cellular level of TS. Greater TS overproduction (80-fold) was obtained by selection for FdUrd resistance in the presence of 10 microM folinic acid and 100 microM deoxyinosine. The latter agents were included to expand the folate pool to ensure adequate levels of cofactor during the selection process. Using cDNA plasmid pMTS-4, which is complementary to mouse TS mRNA, we show that TS overproduction in the HEp-2 variants is accompanied by a 100-fold increase in TS mRNA and a 100-fold amplification of the TS structural gene. Thus, TS overproduction and gene amplification is a mechanism of resistance to FdUrd in human cells.
Mol Pharmacol 1985 Nov
PMID:Thymidylate synthase overproduction and gene amplification in fluorodeoxyuridine-resistant human cells. 293 32

A panel of seven human colorectal cell lines of differing phenotype has been examined to elucidate the role of thymidylate synthase (TS) in the response to 5-fluoro-2'-deoxyuridine (FdUrd). Although TS is a major target of FdUrd, no consistent relationship was observed between the intracellular levels of TS and the response to FdUrd among the cell lines. Levels of thymidine kinase and dihydrofolate reductase, enzymes that are involved in generation of ligands that form the inhibitory ternary complex with TS, do not correlate with FdUrd response. Two cell lines that exhibit innate resistance to FdUrd, relative to the other cell lines, have variations in TS enzyme structure or gene structure. Cell line HCT 116 contains two forms of TS, as defined by isoelectric focusing. One form, which is unique to HCT 116, is more basic than the common form, which is present in all the cell lines. Cell line RCA contains a variation in the TS structural gene, as defined by restriction fragment-length analysis. These structural variations, which are associated with reduced response to FdUrd, may serve as markers for reduced clinical response to TS-directed chemotherapy.
Mol Pharmacol 1988 Oct
PMID:Thymidylate synthase as a determinant of 5-fluoro-2'-deoxyuridine response in human colonic tumor cell lines. 297 71

Inhibition of thymidylate synthase (TS) is an important mechanism of action of fluoropyrimidine antimetabolites. Thus, TS structure and expression are expected to be determinants of response to these agents. The role of TS in fluoropyrimidine response has been analyzed in a panel of human colonic tumor cell lines. Previous work has demonstrated that there is little correlation between TS concentration and sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd) among these cell lines, suggesting that parameters other than the TS levels are responsible for the variations in drug response. One such parameter has been identified in cell line HCT 116. This line, which is relatively resistant to FdUrd, produces two structural forms of TS, as determined by mobility of the enzyme in isoelectric focusing polyacrylamide gels. One form is common to all the cell lines, whereas a variant form, which is more basic and is encoded by a separate structural gene, is unique to HCT 116. Cells expressing one or the other TS form have been isolated and used to demonstrate that the variant form is associated with FdUrd resistance. Kinetic experiments indicate that the variant TS has reduced affinities for 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, which are ligands involved in formation of a stable inhibitory complex with the enzyme. Thus, the innate resistance of cell line HCT 116 to FdUrd is derived, at least in part, from production of an altered structural form of TS having reduced affinity for ligands.
Mol Pharmacol 1988 Oct
PMID:A naturally occurring variation in thymidylate synthase structure is associated with a reduced response to 5-fluoro-2'-deoxyuridine in a human colon tumor cell line. 297 72

We have previously isolated fluorodeoxyuridine-resistant mouse fibroblast (LU3-7) and neuroblastoma (FUdR-R) cell lines that overproduce thymidylate synthase and the mRNA for this enzyme up to 50-fold as compared to the parental cell lines. We have also cloned cDNA corresponding to mouse thymidylate synthase mRNA into pBR322. In the present study, we used this cloned cDNA as a hybridization probe in Southern blot analysis of DNA from the parental and overproducing cell lines. These analyses showed that the thymidylate synthase gene is amplified 50-100 fold in LU3-7 cells and about 30-fold in FUdR-R cells when compared to the respective parental cells. The sizes of the restriction fragments were the same in the parental and overproducing cells of each type, suggesting that extensive rearrangements have not occurred in the vicinity of the thymidylate synthase gene during the amplification process. However, not all of the fragments in the parental cells were amplified in the overproducing cells, suggesting that there may be multiple genes or pseudogenes for the enzyme. Restriction fragment length polymorphisms were detected when analyzing DNA from several different mouse cell lines. When LU3-7 cells were grown in the absence of selective pressure, the level of thymidylate synthase overproduction and the number of copies of the thymidylate synthase gene decreased in parallel.
Mol Pharmacol 1985 Jul
PMID:Thymidylate synthase gene amplification in fluorodeoxyuridine-resistant mouse cell lines. 299 33

We have examined the pattern of transcription exhibited by four genes in the dTTP biosynthetic pathway of Saccharomyces cerevisiae. Consistent with the results reported previously by Storms et al. (1984), the TMP1 (or CDC21) gene encoding thymidylate synthase was found to be transcribed in a periodic manner during the cell cycle with maximal mRNA levels occurring just prior to the onset of DNA replication. Three other genes in this pathway DCD1, DUT1 and DFR1 encoding dCMP deaminase, dUTP pyrophosphatase and dihydrofolate reductase, respectively, exhibited relatively constant levels of transcription throughout the cell cycle. These results, particularly for DFR1, are in marked contrast with those obtained in other eukaryotic systems which have suggested that, in general, genes encoding enzymes involved in DNA precursor synthesis are subject to cell cycle regulation. Thus, periodic transcription is not a property common to all genes involved in DNA replication in this eukaryote.
Mol Gen Genet 1986 Sep
PMID:Transcription of genes encoding enzymes involved in DNA synthesis during the cell cycle of Saccharomyces cerevisiae. 302 Mar 75

5-Fluorouracil, 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorocytidine (FCyd), 5-fluoro-2'-deoxycytidine (FdCyd), 5-trifluoro-2'-deoxythymidine (F3dThd), and the 5'-monophosphates and 3',5'-cyclic monophosphates thereof were found to inhibit thymidine kinase-deficient (TK-) mutant strains of herpes simplex virus (HSV) at a much lower concentration than the wild-type (TK+) HSV strains. Other 5-substituted 2'-deoxyuridines that have previously been recognized as potent thymidylate synthase inhibitors behaved in a similar fashion. The activity of FdUrd, FdCyd, F3dThd, and their 3',5'-cyclic monophosphates against TK-HSV was readily reversed by 2'-deoxythymidine (dThd) but not by 2'-deoxyuridine (dUrd). These compounds also inhibited the incorporation of [6-3H]dUrd into DNA at a concentration which was up to 5 orders of magnitude lower than the concentration at which the incorporation of [methyl-3H] dThd was inhibited. Thus, while not being a target for the well established anti-HSV compounds in TK+HSV-infected cells, thymidylate synthase appears to be an important target in TK-HSV-infected cells. In addition to dTMP synthase, TK-HSV-infected cells appear to reveal other therapeutically exploitable targets such as OMP decarboxylase (towards pyrazofurin), CTP synthase (towards carbodine and its cyclopentenyl analogue), dihydrofolate reductase (towards methotrexate), and S-adenosylhomocysteine hydrolase (towards neplanocins).
Mol Pharmacol 1987 Aug
PMID:Potent activity of 5-fluoro-2'-deoxyuridine and related compounds against thymidine kinase-deficient (TK-) herpes simplex virus: targeted at thymidylate synthase. 303 43

Several new crystal forms of thymidylate synthase (5,10-methlenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) were obtained by controlled pH change. In the crystals the dimeric molecule has a 2-fold symmetry axis coinciding with crystallographic symmetry. The crystals scatter to at least 2.7 A resolution in the synchrotron X-ray beam and appear to be suitable for high-resolution X-ray diffraction analysis. The crystals were successfully derivatized and preliminary results are reported for the covalent inhibitory ternary complex of thymidylate synthase, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.
J Mol Biol 1986 Sep 05
PMID:Crystallization and crystallographic data for new forms of thymidylate synthase from Lactobacillus casei. 309 84

The bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum has been purified to homogeneity using a sequence of separation steps including phenyl-Superose, gel filtration, dye affinity matrix, hydroxyapatite, and anion exchange chromatography. The specific activity of dihydrofolate reductase increased approximately 24,000-fold from 3.3 units mg-1 protein to 79,000 units mg-1 protein after five successive chromatographic steps with a yield of 31%. Both enzyme activities coeluted as a symmetric peak in highly purified preparations, indicating the existence of a bifunctional enzyme complex in P. falciparum. The apparent molecular weight of the native complex was approximately 120,000 as determined by gel filtration. When individual fractions of the anion exchange column were subject to polyacrylamide electrophoresis under denaturing conditions, the increase in intensity of a single band correlated with the amount of both the thymidylate synthase and dihydrofolate reductase activity. Further purification led to an electrophoretically pure protein (yield 2.6%) with an apparent molecular weight of 67,000, suggesting that the bifunctional enzyme complex from P. falciparum is composed of two subunits of identical size and charge.
Mol Pharmacol 1987 Dec
PMID:Purification of the bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum. 332 Jul 42


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