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)

Rapid and quantitative polymerase chain reaction (PCR) assays based upon the competitive template technique have been developed for human dihydrofolate reductase (DHFR; E.C.1.5.1.3) and thymidylate synthase (TS; E.C.2.1.1.45) mRNAs. In various tumor cell lines and clinical tumor biopsies, TS mRNA levels correlated with TS levels as determined by [3H]-fluorodeoxyuridylate binding. Levels of DHFR and TS mRNAs, determined by PCR, correlated with mRNA quantitation by conventional dot blot methodology. The ratio of TS/DHFR mRNAs in a number of human carcinoma cell lines varies from 0.4 to 9.9 but ranges from 1 to greater than 1.5 x 10(3) in a number of tumor samples. Differences in the TS/DHFR mRNA ratio in tumors as compared with cultured cells reflects low levels of DHFR mRNA in some tumors. In patients treated with a combination of 5-fluorouracil and leucovorin, mRNA levels for TS increased approximately an order of magnitude in tumor samples 4 and 24 hr after drug treatment, whereas TS levels decreased. These results have significance for the biochemical pharmacology of antifolates and fluorinated pyrimidines in vivo and the relevance of cell culture models for antifolate chemotherapy and drug resistance.
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PMID:Quantitation of dihydrofolate reductase and thymidylate synthase mRNAs in vivo and in vitro by polymerase chain reaction. 159 83

Methylbenzoprim (MBP) is a potent inhibitor of dihydrofolate reductase, which is more selective for mammalian than bacterial enzymes. Crystal-structure studies on the free base of MBP, with two independent molecules, and the ethanesulfonate salt, have demonstrated three significantly different conformations for MBP. With the MOPAC optimized MBP cation as starting point, the COSMIC energy was monitored as torsion angles were changed in 5 degrees increments. The barrier to rotation about C(5)-C(11) can create two slowly interconverting rotamers, in agreement with NMR studies. Two conformations of the cation that fit the human DHFR structure from the Brookhaven Protein Data Bank have been found. They differ chiefly by a half-turn about C(5)-C(11), positioning the nitro group on opposite sides but allowing the central and benzylic rings to find hydrophobic surroundings. The central ring is close enough to the predicted position of the cofactor NADPH to make competition likely. Kinetic studies with rat liver DHFR show that MBP is an inhibitor that competes with NADPH as well as dihydrofolate.
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PMID:Structural studies on bio-active compounds. 20. Molecular modeling and crystallographic studies on methylbenzoprim, a potent inhibitor of dihydrofolate reductase. 161 54

Three mutations in Plasmodium falciparum yielding increased resistance to pyrimethamine were obtained following treatment with chemical mutagens and selection in presence of pyrimethamine. From parasite clone TM4/8.2 a mutant, TM4/8.2/4.1, was produced which raised pyrimethamine resistance about 500 times and was found to involve an amino acid change in the DHFR-TS enzyme molecule from Ser108 to Asn108. A clone of another isolate, T9/94, yielded a mutant, T9/94/300.300, raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile164 to Met164. However, another mutant from T9/94, T9/94/M1-1(b3), although it raised the pyrimethamine resistance 100 times, did not involve any changes in the coding sequence of the DHFR-TS gene, but resulted in the production of about twice as much DHFR-TS enzyme as the original clone T9/94. No amplification of the DHFR-TS gene was detected. It is concluded that changes in pyrimethamine resistance of malaria parasites may arise in at least 2 ways: (1) by structural changes in the DHFR domain of the DHFR-TS gene (as previously found by other workers); (2) by other changes, possibly affecting the expression of the DHFR-TS gene. The relative importance of these 2 mechanisms in causing resistance in wild populations of P. falciparum is discussed.
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PMID:Pyrimethamine resistant mutations in Plasmodium falciparum. 162 Jan 55

Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.
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PMID:Double-minute chromosomes as megabase cloning vehicles. 162 62

The polymerase chain reaction (PCR) represents an alternative to the current methods for investigating DNA damage and repair in specific genomic segments. In theory, any DNA lesion which blocks Taq polymerase can be measured by this assay. We used quantitative PCR (QPCR) to determine the lesion frequencies produced by cisplatin and ultraviolet light (UV) in a 2.3 kilobase (kb) segment of mitochondrial DNA and a 2.6 kb segment of the DHFR gene in mouse leukemia L1210 cells. The frequency of UV-induced lesions increased linearly with dose, and was 0.58 lesions/10 kb/10 J/m2 in the mitochondrial DNA, and 0.37 lesions/10 kb/10 J/m2 in the DHFR gene. With cisplatin, the lesion frequency also increased linearly with dose, and was 0.17 lesions/10 kb/10 microM in the DHFR gene, and 0.07 lesions/10 kb/10 microM in mitochondrial DNA. This result is contrary to that of Murata et al., 1990 (1), in which mitochondrial DNA received greater cisplatin damage than did nuclear DNA. Using PCR to measure the repair of UV-induced lesions in the DHFR gene segment, we observed that less than 10% of the lesions were removed by 4 h, but over 70% of the lesions were removed by 8 h. Repair of 43% of UV-induced lesions in mitochondrial DNA was also observed during a 24 h period.
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PMID:Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay. 163 Sep 19

4-Nitroquinoline 1-oxide (4NQO) is a model chemical carcinogen that has often been referred to as a UV mimetic agent. Previous studies have indicated that UV-induced pyrimidine dimers are repaired preferentially and strand-specifically in actively transcribing genes. In the current study we have examined the gene-specific and strand-specific repair of 4NQO in Chinese hamster ovary B-11 cells treated with 2.5 microM 4NQO. The methodology used for detecting adducts involved the treatment of DNA from 4NQO-exposed cells with uvrABC excinuclease, which incises DNA at adduct sites, followed by denaturing gel electrophoresis of DNA, Southern hybridization, and probing for the sequence of interest. We examined the active and inactive coding regions of the DHFR gene, the active adenine phosphoribosyltransferase gene, relatively inactive c-fos oncogene, and the mitochondrial genome for 4NQO adducts. Initial 4NQO adduct levels found in these genes varied from 1.10 to 1.52 adducts/10 kilobases. Little difference in repair was found between active coding and inactive regions of the DHFR gene, or between DHFR, adenine phosphoribosyltransferase, and c-fos genes, which are transcribed at different levels. Approximately 71% of 4NQO adducts were repaired within 24 h in all gene sequences examined. During this same time period, approximately 51% of adducts were repaired from the genome overall, as determined by comparing the removal of bound radiolabeled 4NQO to total DNA. The results indicate that 4NQO adducts, unlike UV light-induced cyclobutane pyrimidine dimers (UV dimers), are not preferentially repaired in transcriptionally active genes. However, there may be regions of the genome that are not repaired with the same efficiency as the specific genes examined here. In addition, little to no difference was observed in the repair of 4NQO adducts in the transcribed and nontranscribed strands of the DHFR gene, a finding which is also in contrast to results with UV dimers. Interestingly, 4NQO adducts, unlike UV dimers, were removed from the mitochondrial genome, suggesting that repair of select lesions occurs in this organelle. Thus, there appear to be some differences in the repair pathways operating for 4NQO adducts and UV dimers, particularly with respect to gene- and strand-specific DNA repair.
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PMID:Gene- and strand-specific damage and repair in Chinese hamster ovary cells treated with 4-nitroquinoline 1-oxide. 163 32

In an earlier investigation of the influence of high level expression of p21H-ras, rat-1 cells were co-transfected with a selectable vector (pSV2Neo), an amplifiable vector (encoding dihydrofolate reductase; DHFR) and an H-ras expression vector. In this study we have analyzed the gene dose and expression levels of the three co-transfected plasmid vectors in cell lines that had been selected and isolated at different methotrexate concentrations. Growth of the cells in the absence of selection and Southern blot analyses indicate that the transfected vectors are stably co-integrated into the host genome. High expression levels from all three co-transfected vectors were evident at both the mRNA and protein levels, indicating that they are tightly linked in the host genome. The presence of a large amount of unspliced H-ras mRNA in cells expressing high levels of H-ras p21 indicates that processing of mRNA may be rate-limiting. Comparison of the gene dose and expression levels shows that the resistance of cells to increased methotrexate concentrations can occur by different mechanisms. It is concluded that co-transfection of individual plasmid vectors into rat-1 cells, followed by methotrexate selection, is an effective manner of achieving high level expression of proteins in cultured cells.
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PMID:Analysis of co-transfected expression vectors amplified by methotrexate selection in rat-1 cells. 166 42

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.
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PMID:Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth. 169 88

The role of cytokines in vivo has been difficult to assess. This difficulty is due, in part, to the limited number of producer cells and the strict regulation of cytokine production. In order to address this situation, we have developed assays which allow us to quantitate both protein production and steady state mRNA levels from specific in vivo sites. In this report, we present data utilizing these assays on cells obtained from draining LN following specific sensitization with antigen in vivo. In order to determine the relative quantities of cytokine mRNA, we modified the reverse transcriptase-polymerase chain reaction which had been previously described. The modified assay is (1) linear over a large concn range of input template (2) demonstrates a high degree of reproducibility (SE approximately 13%) and (3) is very sensitive. Utilizing this assay, we have measured a constitutive mRNA (DHFR), quantitated both the presence of lymphokine mRNA (IL-2) and the induction of cytokine mRNA (TNF alpha). In this report we have examined the kinetics of TNF alpha mRNA expression and have demonstrated that following epicutaneous sensitization with picryl chloride, there is rapid induction (within 24 hr) of TNF alpha mRNA in the draining LN and that the levels of mRNA remain detectable through d7. In addition, we determined the time course of production of TNF protein by the draining LN cells and found that it was similar to that of the mRNA levels. A potential pathologic role for immune response generated TNF alpha is also discussed. We believe these experiments demonstrate that cytokine production following antigen-specific sensitization in vivo can be analyzed at both the cellular and molecular level. The data suggests that this approach can be used to study cytokine regulation in vivo.
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PMID:Quantitation of cytokine mRNA levels utilizing the reverse transcriptase-polymerase chain reaction following primary antigen-specific sensitization in vivo--I. Verification of linearity, reproducibility and specificity. 171 71

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer. Mutagenesis of the protease-sensitive region (residues 430-440), which is part of the crystallized domain, indicates that any alteration of the wild-type sequence results in increased proteolysis of the p66 subunit. A model of asymmetric processing in HIV-1 reserve transcriptase which involves partial unfolding of the RNase H domain is proposed based on these results and the recently reported three-dimensional structure of this domain.
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PMID:Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase. 171 88


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