Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

High speed centrifugal supernatant fractions of homogenates of a number of trypanosomatids were assayed for thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) activity using the method of Lomax and Greenberg (1967) J. Biol. Chem. 242, 109-113). Similar activities were detected in Crithidia fasciculata, Crithidia oncopelti, the blood forms of Trypanosoma brucei, Trypansoma congolense and Trypanosoma lewisi and the blood, intracellular and culture forms of Trypanosoma cruzi, suggesting that all species synthesize at least some thymidylate de novo. The properties of the activities in C. fasciculata and the three forms of T. cruzi were compared with those of the isofunctional bacterial and mammalian enzymes. The trypanosotamid enzyme was inhibited by Mg2+, was much more sensitive to mercaptoethanol, had higher apparent Km values for substrate (dUMP) and cofactor (tetrahydrofolate), had a higher apparent molecular weight and was markedly more sensitive to inhibition by suramin. It is, therefore a possible target for chemotherapeutic attack, either on its own or in combination with a dihydrofolate reductase inhibitor. No evidence was obtained for the regulation of the trypanosomatid enzyme, either by its product, dTMP, or by dTDP or dTTp. This result agrees with previous studies which suggested that in trypanosomatids, the level of dTMP was regulated, at least in part, by a catabolic pathway consisting of a thymidylate phosphatase and a thymidine phosphorylase which degraded the excess of dTMP to thymine.
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PMID:Presence and properties of thymidylate synthase in trypanosomatids. 1 96

Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5' cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.
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PMID:Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues. 144 68

The repression of MetE synthesis in Escherichia coli by vitamin B12 is known to require the MetH holoenzyme (B12-dependent methyltransferase) and the metF gene product. Experiments using trimethoprim, an inhibitor of dihydrofolate reductase, show that the MetF protein is not directly involved in the repression, but that N5-methyltetrahydrofolic acid (N5-methyl-H4-folate), the product of the MetF enzymatic reaction is required. Since the methyl group from N5-methyl-H4-folate is normally transferred to the MetH holoenzyme to form a methyl-B12 enzyme, the present results suggest that a methyl-B12 enzyme is involved in the vitamin B12 repression of metE expression. Other results argue against the possibility that a methyl-B12 enzyme functions in this repression solely by decreasing the cellular level of homocysteine, which is required for MetR activation of metE expression. Experiments with metJ mutants show that the MetJ protein mediates about 50% of the repression of metE expression by B12 but is totally responsible for the regulation of metF expression by vitamin B12.
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PMID:Role of the metF and metJ genes on the vitamin B12 regulation of methionine gene expression: involvement of N5-methyltetrahydrofolic acid. 173 76

Cycloguanil, the active metabolite of the antimalarial drug proguanil, is an inhibitor of dihydrofolate reductase as is another antimalarial, pyrimethamine. Its use has been limited by the rapid development of resistance by parasites around the world. We have determined the cycloguanil- and pyrimethamine-sensitivity status of 10 isolates of Plasmodium falciparum and have sequenced in all these isolates the dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) portion of the DHFR-thymidylate synthase (TS; 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) gene. Instead of the known serine-to-asparagine change at position 108 that is important in pyrimethamine resistance, a serine-to-threonine change at the same position is found in cycloguanil-resistant isolates along with an alanine-to-valine change at position 16. We conclude that pyrimethamine and cycloguanil resistance most commonly involve alternative mutations at the same site. However, we also have identified a parasite with a unique set of changes that results in resistance to both drugs.
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PMID:Amino acids in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum involved in cycloguanil resistance differ from those involved in pyrimethamine resistance. 218 21

A T7 RNA transcript coding for mouse dihydrofolate reductase (DHFR) was utilized as a substrate for the N6-methyladenosine mRNA methyltransferase isolated from HeLa cell nuclei. This transcript acted as a 3 fold better substrate than either prolactin mRNA or a synthetic RNA substrate which contained multiple methylation consensus sequences. Formation of internal N6-methyladenine (m6A) residues in the DHFR transcript was shown to increase slightly by the absence of a 7-methylguanine-2'-O-methyl cap structure. Using T7 transcripts from different regions of the DHFR gene, the majority of the m6A residues were localized to the coding region and a segment of the transcript just 3' to the coding region. This data suggests that DHFR mRNA contains multiple methylation sites with most of these sites concentrated in the coding region of the transcript.
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PMID:Analysis and in vitro localization of internal methylated adenine residues in dihydrofolate reductase mRNA. 239 44

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.
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PMID:Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases. 265

The DNA sequences of the diadenosine tetraphosphatase gene (apaH) and of the flanking regions were determined. Three other genes were identified in the flanking regions: ksgA, apaG and folA encoding, respectively, a 16 S rRNA methyltransferase, an unidentified protein of Mr 13,826 and dihydrofolate reductase, with the order folA-apaH-apaG-ksgA. The apaH gene is thus located between folA and ksgA at 1 min on the Escherichia coli chromosome linkage map and folA is transcribed clockwise, whereas ksgA, apaG and apaH are transcribed in the opposite direction. It was shown that ksgA, apaG and apaH can be expressed from a polycistronic mRNA originating from a promoter (p1) located upstream of ksgA. However, another promoter (p2) was found within the ksgA structural gene. This promoter, active in vivo, can account for p1-independent expression of the two distal cistrons, apaG and apaH. Finally, the effect on diadenosine tetraphosphatase over-production of a frameshift mutation causing premature translational termination of apaG suggests that expression of apaG and apaH is coupled at the translational level.
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PMID:The gene for Escherichia coli diadenosine tetraphosphatase is located immediately clockwise to folA and forms an operon with ksgA. 303 29

Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolated:dUMP C-methyltransferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and poly(gamma-glutamyl) conjugates of pteroate and methotrexate (MTX) has been examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(gamma-glutamate)s within 4 hr of exposure to 1 microM MTX. MTX and MTX(gamma-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and I-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to two sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(gamma-glutamate), 7,8-dihydropteroylpentaglutamate, and I-5-10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, < 10, and < 10 nM, respectively, by equilibrium dialysis. We propose that the sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.
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PMID:5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates. 616 May 78

The molecular weight of dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) from protozoa has been reported to be 5- to 10-fold larger than the isofunctional enzyme of most other organisms studied, based on gel filtration. This enzyme from the protozoal flagellate Crithidia fasciculata has been purified to homogeneity and found to be a bifunctional protein with thymidylate synthase (5,10-methylene tetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) activity. The purified protein, eluted from methotrexate-Sepharose columns by dihydrofolate, migrated as a single band on both nondenaturing and denaturing polyacrylamide gel electrophoresis. The monomer Mr is 56,700 +/- 200. The native Mr was calculated to be 107,000 from a sedimentation coefficient of 5.9 and Stokes radius of 4.4 nm. Dihydrofolate reductase and thymidylate synthase activities of the rodent malaria organism Plasmodium berghei also copurified on Sephadex G-200 and methotexate-Sepharose columns, suggesting that this unique bifunctional protein might occur throughout the Protozoa.
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PMID:Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. 693 11

In vivo selection of hematopoietic stem cells (HSCs) offers an approach to enrichment of genetically modified blood cells in the context of gene therapy for blood disorders. We have previously demonstrated efficient HSC selection in mice using retroviral vectors expressing dihydrofolate reductase (DHFR) or methylguanine methyltransferase (MGMT) drug resistance genes. In this study, we examined whether drug selection was followed by subsequent HSC regeneration and, if not, whether regeneration could be augmented by enforced expression of HOXB4, which has previously been shown to enhance HSC regeneration after transplant. Using a murine competitive repopulation model, we found that selection using either the DHFR or the MGMT system was accompanied by a significant overall reduction in repopulating activity in secondary transplant assays, although hematopoiesis remained normal after recovery. Inclusion of a HOXB4 expression cassette in the DHFR vector resulted in a partial restoration of HSC numbers following selection and was associated with an increase in HSC selection efficiency. These results illustrate that while drug resistance vectors can protect transduced HSC from cytotoxic drugs, the self-renewal capacity of transduced HSCs is limited following in vivo selection. Strategies that increase self-renewal capacity could increase the efficiency and safety of in vivo selection.
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PMID:Reduction in hematopoietic stem cell numbers with in vivo drug selection can be partially abrogated by HOXB4 gene expression. 1294 10


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