Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The dihydrofolate reductase amplicon in methotrexate-resistant mosquito cells provides an amplified gene in insects that can be compared directly to the corresponding amplified locus in mammalian cells. A cloned Aedes albopictus dihydrofolate reductase gene was used as a probe to examine the structure of dihydrofolate reductase alleles in sensitive and resistant cells. In wild type cells, two distinct alleles could be distinguished by restriction fragment length polymorphisms, one of which was amplified in methotrexate-resistant cells. Subsequent to amplification, an additional polymorphism at a ten base-pair XmnI recognition site indicated that the amplified mosquito gene is subject to genetic changes similar to those that have been described in amplified genes from mammalian cells. Pulsed-field gel electrophoresis was used to determine that the minimum size of the mosquito dihydrofolate reductase amplicon was 140 kilobases; ethidium bromide staining patterns suggested a size of at least 233 kilobases. Dihydrofolate reductase probes hybridized to distinct locations in five of the thirteen chromosomes in Mtx-5011-128 cells, indicating that the amplified DNA probably occurs as tandem direct or inverted repeats.
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PMID:An amplified mosquito dihydrofolate reductase gene: amplicon size and chromosomal distribution. 908 53

Dihydrofolate reductase was synthesized in a batch system in the presence of the affinity ligand methotrexate, bound to various matrices. Two types of gel were used: commercial methotrexate-agarose with pores inaccessible for translation machinery and methotrexate-POROS with pores easily accessible for translation reaction mixture components. The transcription/translation reaction was not inhibited by either the immobilized methotrexate or the matrix. The enzyme was synthesized with a high yield and could simultaneously be removed from the reaction mixture by the affinity matrix during the synthesis. With methotrexate-POROS present the reaction probably proceeded mainly in the pores of the gel. Kinetic limitations to the reaction in the presence of the gel were not observed. Active dihydrofolate reductase was eluted from methotrexate-POROS. The activity recovered was higher than dihydrofolate reductase activity synthesized in free solution system. The influence of the presence of immobilized methotrexate on dihydrofolate reductase synthesis will be further studied in a novel type of a continuous protein synthesis system.
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PMID:Dihydrofolate reductase synthesis in the presence of immobilized methotrexate. An approach to a continuous cell-free protein synthesis system. 917 38

Previous studies suggest that dihydrofolate reductase (DHFR) regulates its own translation. Moreover, intracellular levels of DHFR protein increase following exposure to the antifolate methotrexate (MTX), suggesting that MTX may release the translational inhibition mediated by DHFR [Chu et al. (1993) Biochemistry 32,4756-4760; Ercikan et al. (1993) Adv. Exp. Med. Biol. 338, 537-540]. To further investigate the role of DHFR in translational autoregulation, we have considered the possibility that DHFR directly contacts its cognate mRNA. Binding studies using a series of truncated DHFR mRNAs as probes localized the DHFR/RNA interaction to a 100-base-pair region containing two putative stem-loop structures; initial studies indicated that both of these loop structures are involved in protein binding. Moreover, the binding of MTX to DHFR prevents interaction of the protein with its cognate mRNA, thereby relieving translational autoregulation.
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PMID:Dihydrofolate reductase protein inhibits its own translation by binding to dihydrofolate reductase mRNA sequences within the coding region. 931 71

Dihydrofolate reductase activity is required for many biosynthetic pathways including nucleotide synthesis. Its expression is therefore central to cellular growth, and it has become a key target for cancer chemotherapy. Transcription of the dihydrofolate reductase gene is regulated with growth, being expressed maximally in late G1/early S phase following serum stimulation of quiescent cells. This regulation is directed by a promoter which contains binding sites for only the transcription factors Sp1 and E2F. In this study, the role of these promoter elements in growth/cell cycle regulation of dihydrofolate transcription was addressed directly by transient transfection of Balb/c 3T3 cells with mutant promoter-reporter gene constructs. The E2F sites were found to repress transcription in G0 and early G1 but did not contribute to the level of transcription in late G1/S phase. In contrast, Sp1 sites were able to mediate induction of transcription from the dihydrofolate reductase promoter, as well as a heterologous promoter, following serum stimulation of quiescent cells. These findings add dihydrofolate reductase to a growing list of genes at which E2F sites are primarily repressive elements and delineate a role for Sp1 sites in the growth/cell cycle regulation of transcription.
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PMID:Distinct roles for Sp1 and E2F sites in the growth/cell cycle regulation of the DHFR promoter. 932 36

Dihydrofolate reductase is an essential bacterial enzyme necessary for the maintenance of intracellular folate pools in a biochemically active reduced state. In this report, the Mycobacterium avium folA gene was identified by functional genetic complementation, sequenced, and expressed for the first time. It has an open reading frame of 543 bp with a G + C content of 73%. The translated polypeptide sequence shows 58% identity to the consensus sequence of the conserved regions from eight other bacterial dihydrofolate reductases. Recombinant M. avium dihydrofolate reductase was expressed actively in Escherichia coli, and SDS-PAGE analysis revealed a 20 kDa species, agreeable with that predicted from the polypeptide sequence:
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PMID:Identification and cloning of the Mycobacterium avium folA gene, required for dihydrofolate reductase activity. 936 62

Site-directed incorporation of the amino acid analogue p-fluoro-phenylalanine (p-F-Phe) was achieved in Escherichia coli. A yeast suppressor tRNA(Phe)amber/phenylalanyl-tRNA synthetase pair was expressed in an analogue-resistant E. coli strain to direct analogue incorporation at a programmed amber stop codon in the DHFR marker protein. The programmed position was translated to 64-75% as p-F-Phe and the remainder as phenylalanine and lysine. Depending on the expression conditions, the p-F-Phe incorporation was 11-21-fold higher at the programmed position than the background incorporation at phenylalanine codons, showing high specificity of analogue incorporation. Protein expression yields of 8-12 mg/L of culture, corresponding to about two thirds of the expression level of the wild-type DHFR protein, are sufficient to provide fluorinated proteins suitable for 19F-NMR spectroscopy and other sample-intensive methods. The use of a nonessential "21st" tRNA/synthetase pair will permit incorporation of a wide range of analogues, once the synthetase specificity has been modified accordingly.
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PMID:Expansion of the genetic code: site-directed p-fluoro-phenylalanine incorporation in Escherichia coli. 952 Nov 19

If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro. We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our first attempt, a sequence encoding either an active or an inactive ribozyme was connected upstream of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP). However, a critical defect may be associated with introduction of a ribozyme upstream of the DHFR gene because, during actual screening for active ribozymes on the 5' side from a pool of random sequences, there is the danger of selecting sequences that are not related to the activity of ribozymes. Indeed, some upstream linker sequences affected the level of expression of the DHFR protein and, as a result, the resistance of Escherichia coli to TMP. Therefore, we newly constructed a 3'-connected ribozyme system, and activities in vivo of 5'-connected and 3'-connected ribozymes were compared. We found that the cleavage efficiencies in vivo were nearly identical for the two types of ribozyme, 24% for the 5'-side ribozyme and 23% for the 3'-side ribozyme, indicating that polysomes did not seem to inhibit the action of the 3'-connected ribozyme. In both cases, when cells were transformed with a 1 : 1 mixture of active and inactive ribozyme-coding plasmids, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP.
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PMID:Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. 953 62

Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.
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PMID:Dihydrofolate reductase: a potential drug target in trypanosomes and leishmania. 974 68

To develop a gene therapy protocol suitable for the treatment of a benign disease such as Gaucher disease, we have developed two bicistronic vectors that allow transduced cells to be selected for with methotrexate (MTX). The two vectors differ in the presence or absence of a mutant polyoma enhancer (delta Mo + PyF101) replacing the wild-type retroviral enhancer in the LTR. Infection of human TF-1 and K562 cells, Gaucher type II fibroblasts and murine hemopoietic bone marrow cells conferred MTX resistance and glucocerebrosidase (GC) expression. Upon increasing MTX concentrations, the number of proviral copies and GC activity increased, demonstrating in vitro selection of retrovirus-transduced cells. At high MTX selection pressure, up to 140 microM for infected Gaucher type II fibroblasts, no endogenous wild-type DHFR amplification could be detected, indicating that both retroviral constructs provide sufficient DHFR protein levels. Upon transduction, murine bone marrow cells were protected against otherwise lethal MTX concentrations (range 1-5 microM MTX). Flow cytometry specific for human GC (hGC) demonstrated that in vitro selection resulted in increased percentages of hGC-positive murine cells. In conclusion, the generated bicistronic vectors are ideally suited to investigate whether an in vivo selection approach for retrovirus-transduced cells is feasible. Such a strategy might abolish the need for a high initial transduction efficiency and might result in a gene therapy protocol devoid of the undesirable need for marrow ablative treatment of the recipient.
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PMID:Methotrexate selectable retroviral vectors for Gaucher disease. 993 Mar 44

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate and is essential for the synthesis of thymidylate, purines and several amino acids. Inhibition of the enzyme's activity leads to arrest of DNA synthesis and cell death. The enzyme has been studied extensively as a drug target for bacterial, protozoal and fungal infections, and also for neoplastic and autoimmune diseases. Here, we report the crystal structure of dihydrofolate reductase from Mycobacterium tuberculosis, a human pathogen responsible for the death of millions of human beings per year. Three crystal structures of ternary complexes of M. tuberculosis DHFR with NADP and different inhibitors have been determined, as well as the binary complex with NADP, with resolutions ranging from 1.7 to 2.0 A. The three DHFR inhibitors are the anticancer drug methotrexate, the antimicrobial trimethoprim and Br-WR99210, an analogue of the antimalarial agent WR99210. Structural comparison of these complexes with human dihydrofolate reductase indicates that the overall protein folds are similar, despite only 26 % sequence identity, but that the environments of both NADP and of the inhibitors contain interesting differences between the enzymes from host and pathogen. Specifically, residues Ala101 and Leu102 near the N6 of NADP are distinctly more hydrophobic in the M. tuberculosis than in the human enzyme. Another striking difference occurs in a region near atoms N1 and N8 of methotrexate, which is also near atom N1 of trimethoprim, and near the N1 and two methyl groups of Br-WR99210. A glycerol molecule binds here in a pocket of the M. tuberculosis DHFR:MTX complex, while this pocket is essentially filled with hydrophobic side-chains in the human enzyme. These differences between the enzymes from pathogen and host provide opportunities for designing new selective inhibitors of M. tuberculosis DHFR.
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PMID:Three-dimensional structure of M. tuberculosis dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs. 1062 28


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