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

mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5' untranslated regions (5' UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3' UTR but none in its 5' UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3' UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycle-dependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts.
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PMID:Sequence elements in both the intergenic space and the 3' untranslated region of the Crithidia fasciculata KAP3 gene are required for cell cycle regulation of KAP3 mRNA. 1291 86

Antifolate drugs that target the biosynthesis and processing of essential folate cofactors are widely used for treatment of chloroquine-resistant falciparum malaria. Salvage of pre-formed folate can strongly compromise the efficacy of these drugs in vitro and the availability of folate from the human host in natural infections also influences therapeutic outcomes. To investigate how different parasite lines respond to the presence of exogenous folate, we measured the effect of the latter on the susceptibility of parasites to sulfa-drug blockage of folate biosynthesis, utilising the parents and 22 progeny of the HB3-Dd2 genetic cross of Plasmodium falciparum, together with selected unrelated lines. Complete linkage of the folate utilisation phenotype was observed to a DNA sequence of 48.6 kb lying between nucleotide positions 738,489 and 787,058 of chromosome 4 and encompassing the dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene locus. Examination of the putative ORFs on this fragment upstream (3) and downstream (4) of dhfr-ts revealed no plausible candidate genes for folate processing. Similarly, a marked heterogeneity in the 5'-UTR regions of Dd2 and HB3, manifest as a directly repeated 256 bp sequence in the former, also did not correlate with the folate utilisation phenotype nor apparently influence levels of dhfr-ts transcripts or protein products. By contrast, the nature of the coding sequence of the dhfr domain appeared to play a direct role, with the single mutant (S108N) HB3-type utilising folic acid much less efficiently than other allelic variants. We also compared the processing of exogenous folic acid, folinic acid and p-aminobenzoic acid (pABA) in metabolic labelling studies of HB3 and Dd2. These support the view that DHFR is likely to have a low-level folate reductase activity as well as its normal function of reducing dihydrofolate to tetrahydrofolate, and that a significant hurdle in the utilisation of exogenous folic acid is the initial reduction of fully oxidised folic acid to dihydrofolate, an activity that the single mutant enzyme found in HB3 is postulated to perform particularly poorly. This would mirror earlier studies indicating that the DHFR activity of HB3 is also compromised relative to other variants.
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PMID:Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum. 1528 89

The Crt1 (RFX1) protein in Saccharomyces cerevisiae is an effector of the DNA damage checkpoint pathway. It recognizes a 13-bp cis-regulatory element in the 5'-untranslated region (5'-UTR) of the ribonucleotide reductase genes RNR2, RNR3, and RNR4; the HUG1 gene; and itself. We calculated the weight matrix representing the Crt1p binding site motif according to analysis of the 5'-UTR sequences of the genes that are under its regulation. We subsequently searched the 5'-UTR sequences of all the genes in the yeast genome for the occurrence of this motif. The motif was found in regulatory regions of 30 genes. A statistical analysis showed that it is unlikely that a random gene cluster contains the motif conserved as well as the Crt1p binding site. Analysis of microarray data provided supporting evidence for five putative Crt1p targets: FSH3, YLR345W, UBC5, NDE2, and NTH2. We used reverse transcription-PCR to compare the expression levels of these genes in wild-type and crt1Delta strains. Our results indicated that FSH3, YLR345W, and NTH2 are indeed under the regulation of Crt1p. Sequence analysis of the FSH3p indicated that this protein may be involved in folate metabolism either by carrying serine hydrolase activity required for the novel metabolic pathway involving dihydrofolate reductase (DHFR) or by directly interacting with the DHFR enzyme. We postulate that Crt1p may influence deoxyribonucleotide synthesis not only by regulating expression of the RNR genes but also by modulating DHFR activity. FSH3p shares significant sequence similarity with the product of the human tumor suppressor gene OVCA2. YLR345Wp and NTH2p are enzymes involved in the central metabolism under stress conditions.
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PMID:Identification of new genes regulated by the Crt1 transcription factor, an effector of the DNA damage checkpoint pathway in Saccharomyces cerevisiae. 1549 96

Alternative polyadenylation leads to mRNAs with variable 3' ends. Since a 3'-untranslated region (3'-UTR) often contains cis elements that impact stability or localization of mRNA or translation, selection of poly(A) sites in a 3'-UTR is regulated in mammalian cells. However, the molecular basis for alternative poly(A) site selection within a 3'-UTR has been unclear. Here we show involvement of cleavage factor Im (CFIm) in poly(A) site selection within a 3'-UTR. CFIm is a heterodimeric 3' end-processing complex, which functions to assemble other processing factors on pre-mRNA in vitro. We knocked down 25 kDa subunit of CFIm (CFIm25) in HeLa cells and analyzed alternative poly(A) site selection of TIMP-2, syndecan2, ERCC6 and DHFR genes by northern blotting. We observed changes in the distribution of mRNAs in CFIm25 depleted cells, suggesting a role for CFIm in alternative poly(A) site selection. Furthermore, tissue specific analysis demonstrated that the CFIm25 gene gave rise to 1.1, 2.0 and 4.6 kb mRNAs. The 4.6 kb mRNA was ubiquitously expressed, while the 1.1 and 2.0 kb mRNAs were expressed in a tissue specific manner. We found three likely poly(A) sites in the CFIm25 3'-UTR, suggesting alternative polyadenylation. Our results indicate that alternative poly(A) site selection is a well-regulated process in vivo.
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PMID:Knock-down of 25 kDa subunit of cleavage factor Im in Hela cells alters alternative polyadenylation within 3'-UTRs. 1709 38

The dihydrofolate reductase (DHFR) enzyme is important for folate availability, folate turnover and DNA synthesis. The 19-bp deletion in intron-1 of DHFR has been associated with the risk of having spina bifida affected offspring, supposedly by changing DHFR gene expression. A 9-bp repeat in exon 1 of the mutS homolog 3 (MSH3) gene was recently demonstrated to be also located in the 5'UTR of DHFR and may possibly affect DHFR gene expression as well. We examined the association between these DHFR variants and spina bifida risk and investigated their effect on DHFR expression. Our study population, consisting of 121 mothers of a spina bifida affected child, 109 spina bifida patients, 292 control women and 234 pediatric controls was screened for the DHFR 19-bp deletion and the DHFR 9-bp repeat. DHFR gene expression was measured in 66 spina bifida patients, using real-time PCR analysis. In this study population, the DHFR 19-bp del/del genotype was not associated with spina bifida risk in mothers and children (OR: 0.8; 95%CI: 0.4-1.5 and OR: 1.2; 95%CI: 0.6-2.2, respectively) and both the WT/del and the del/del genotype did not affect DHFR expression relative to the WT/WT genotype (relative expression=0.89, p=0.46 and relative expression=1.26, p=0.24, respectively). The DHFR 9-bp repeat was not associated with spina bifida risk in mothers and children. DHFR expression of the 6/6 allele was 73% increased compared to the 3/3 allele, although not significantly (relative expression=1.73, p=0.09). We did not find evidence for an effect of the DHFR 19-bp deletion or 9-bp repeat on spina bifida risk in mothers and children. An effect of the 6/6 repeat genotype on DHFR expression cannot be ruled out.
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PMID:Variation and expression of dihydrofolate reductase (DHFR) in relation to spina bifida. 1733 64

MicroRNAs are predicted to regulate approximately 30% of all human genes by targeting sequences in their 3' UTR. Polymorphisms in 3' UTR of several genes have been reported to affect gene expression, but the mechanism is not fully understood. Here, we demonstrate that 829C-->T, a naturally occurring SNP, near the miR-24 binding site in the 3' UTR of human dihydrofolate reductase (DHFR) affects DHFR expression by interfering with miR-24 function, resulting in DHFR overexpression and methotrexate resistance. miR-24 has a conserved binding site in DHFR 3' UTR. DHFR with WT and 3' UTR containing the 829C-->T mutation were expressed in DG44 cells that lack DHFR. Overexpression of miR-24 in cells with WT DHFR resulted in down-regulation of DHFR protein, whereas no effect on DHFR protein expression was observed in the mutant 3' UTR-expressing cells. Inhibition of endogenous miR-24 with a specific inhibitor led to up-regulation of DHFR in WT and not in mutant cells. Cells with the mutant 3' UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells. SNP-829C-->T, therefore, leads to a decrease in microRNA binding leading to overexpression of its target and results in resistance to methotrexate. We demonstrate that a naturally occurring miRSNP (a SNP located at or near a microRNA binding site in 3' UTR of the target gene or in a microRNA) is associated with enzyme overproduction and drug resistance.
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PMID:A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance. 1768 70

MicroRNAs are evolutionarily conserved small non-coding RNAs known to inhibit the translation of proteins by binding to the target transcript in the 3' untranslated region. Functional polymorphisms in 3' UTRs of several genes have been reported to be associated with diseases by affecting gene expression. The mechanism by which these polymorphisms affect gene expression and induce variability in a cell is not well understood. It has been suggested that these polymorphisms may interfere with regulatory elements that bind to untranslated region of a gene. Recently, a novel class of functional polymorphisms termed miRSNPs/polymorphisms was reported. defined as a polymorphism present at or near a microRNA binding sites of functional genes that can affect gene expression by interfering with a miRNA function. The work elucidated the mechanism of a functional miRSNP 829C-->T present in 3' UTR of dihydrofolate reductase, an important drug target. The SNP interferes with the miR24 microRNA function and leads to DHFR over expression and methotrexate resistance. In this article we highlight the importance of these miRSNPs or miR-polymorphisms in gene regulation and the mechanism by which these miRSNPs can induce variability in the SNP expressing mutant cell by using drug resistance as an example.
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PMID:MiRSNPs or MiR-polymorphisms, new players in microRNA mediated regulation of the cell: Introducing microRNA pharmacogenomics. 1841 50

Genetic factors are thought to play a role in resistance towards chemotherapeutic agents such as 5-fluorouracil (5-FU). Approximately 30 genes are directly or indirectly involved in 5-FU metabolism, and genetic variation in any of these may contribute to anti-tumor response. Polymorphisms in these genes were analyzed in relation to tumoral mRNA levels of 5-FU metabolizing genes, response to chemotherapy and survival. A total of 21 genetic variants were studied in 35 breast cancer patients treated with 5-FluoroUracil, mitomycin (FUMI) and in a similar cohort of 90 doxorubicin treated breast cancer patients. Genotype distributions were compared using 109 healthy controls. No significant association was found between any polymorphisms and response to chemotherapy as measured by shrinkage of tumor. However, carriers of 3 copies of the TYMS 5'UTR repeat had shorter survival than noncarriers (p = .048) in the FUMI treatment group, but not in the doxorubicin treated group. Carriers of 3 copies of the repeat were also more frequently observed in both patients groups than in healthy controls (p = .034). Several highly significant associations were observed between genotypes and expression levels of 5-FU metabolizing genes. A SNP in codon 72 of TP53 was revealed to be a key regulator of 5-FU metabolizing genes such as DHFR and MTHFR, constituting 50% of all significant associations observed after FUMI therapy. These data suggest that 3 copies of the TYMS 5'UTR repeat may give a treatment specific reduced survival in breast cancer patients, and that TP53 may have a direct, allele specific, role in 5-FU mediated response.
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PMID:Pathway based analysis of SNPs with relevance to 5-FU therapy: relation to intratumoral mRNA expression and survival. 1849 33

In this study, single nucleotide polymorphisms (SNPs) involved in homocysteine metabolism such as CT replacement in the 677th nucleotide in 5,10-methylenetetrahydrofolate reductase (MTHFR) enzyme; 68-bp insertion in the 844th nucleotide of cystathionine beta-synthase (CBS) enzyme; 6-bp insertion/deletion in the region of 3'UTR in thymidylate synthase (TYMS) enzyme and 19-bp deletion in dihydrofolate reductase (DHFR) enzyme were investigated. The effects of these mutations on homocysteine levels were studied. As a result; we found that TT genotype of MTHFR 677 CT is an influencing factor on homocysteine levels in Turkish population. Furthermore, there seems to be another MTHFR 677 TT haplotype, which does not have an effect on homocysteine levels. Our data revealed that other SNPs did not have any influence on homocysteine levels.
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PMID:Single nucleotide polymorphisms that affect homocysteine levels in Turkish population. 1879 60

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3' UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.
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PMID:MiR-24 tumor suppressor activity is regulated independent of p53 and through a target site polymorphism. 2004 Nov 60


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