Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of the inhibition of dihydrofolate reductase by methotrexate on the cellular folates involved in de novo purine and thymidylate biosynthesis has been measured in H35 hepatoma cells grown in 4 microM folic acid or 20 nM folinic acid. The major cellular folate species in cells from medium with folate or folinate is 10-formyltetrahydrofolate (approximately 5 microM), with lesser amounts of 5,10-methylenetetrahydrofolate and tetrahydrofolate. Cultures were exposed to a pulse dose of methotrexate, resulting in the accumulation of nearly exclusively methotrexate polyglutamates (predominantly Glu3, Glu4, and Glu5), or a continuous exposure to the poorly glutamylated analog threo-4-fluoromethotrexate, resulting in 93% intracellular monoglutamate. At 4 hr and 18 hr after exposure to either compound there was extensive depletion of the reduced folate coenzymes, which generally corresponded to the extent of inhibition of glycine and deoxyuridine incorporation. This was accompanied by an increase of the cellular dihydrofolate and 10-formyldihydrofolate. In the H35 cells the effect of methotrexate polyglutamates on the reduced folate coenzyme pools was restricted to dividing cultures, because the reduced folate coenzymes were not depleted in confluent cultures. The results demonstrate that the methotrexate and methotrexate polyglutamates that initially accumulate within dividing H35 cells readily inhibit dihydrofolate reductase but are not adequate to inhibit thymidylate synthase and prevent the depletion of reduced folate coenzymes. Thus, inhibition of de novo glycine and deoxyuridine incorporation into DNA as a result of dihydrofolate reductase inhibitors appears to be closely related to a reduction in the intracellular concentration of 10-formyltetrahydrofolate and 5,10-methylenetetrahydrofolate, the respective folate coenzymes for de novo purine and thymidylate synthesis.
Mol Pharmacol 1992 Nov
PMID:Depletion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate by methotrexate in cultured hepatoma cells. 143 54

Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and beta-glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.
Plant Mol Biol 1992 Dec
PMID:New plant binary vectors with selectable markers located proximal to the left T-DNA border. 146 55

Dihydroxyacetone synthase (DAS) and methanol oxidase (MOX) are the major enzyme constituents of the peroxisomal matrix in the methylotrophic yeast Hansenula polymorpha when grown on methanol as a sole carbon source. In order to characterize their topogenic signals the localization of truncated polypeptides and hybrid proteins was analysed in transformed yeast cells by subcellular fractionation and electron microscopy. The C-terminal part of DAS, when fused to the bacterial beta-lactamase or mouse dihydrofolate reductase, directed these hybrid polypeptides to the peroxisome compartment. The targeting signal was further delimited to the extreme C-terminus, comprising the sequence N-K-L-COOH, similar to the recently identified and widely distributed peroxisomal targeting signal (PTS) S-K-L-COOH in firefly luciferase. By an identical approach, the extreme C-terminus of MOX, comprising the tripeptide A-R-F-COOH, was shown to be the PTS of this protein. Furthermore, on fusion of a C-terminal sequence from firefly luciferase including the PTS, beta-lactamase was also imported into the peroxisomes of H. polymorpha. We conclude that, besides the conserved PTS (or described variants), other amino acid sequences with this function have evolved in nature.
Mol Gen Genet 1992 Nov
PMID:Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha. 146 1

The discriminator nucleotide (position 73) in tRNA has long been thought to play a role in tRNA identity as it is the only variable single-stranded nucleotide that is found near the site of aminoacylation. For this reason, a complete mutagenic analysis of the discriminator in three Escherichia coli amber suppressor tRNA backgrounds was undertaken; supE and supE-G1C72 glutamine tRNAs, gluA glutamate tRNA and supF tyrosine tRNA. The effect of mutation of the discriminator base on the identity of these tRNAs in vivo was assayed by N-terminal protein sequencing of E. coli dihydrofolate reductase, which is the product of suppression by the mutated amber suppressors, and confirmed by amino acid specific suppression experiments. In addition, suppressor efficiency assays were used to estimate the efficiency of aminoacylation in vivo. Our results indicate that the supE glutamine tRNA context can tolerate multiple mutations (including mutation of the discriminator and first base-pair) and still remain predominantly glutamine-accepting. Discriminator mutants of gluA glutamate tRNA exhibit increased and altered specificity probably due to the reduced ability of other synthetases to compete with glutamyl-tRNA synthetase. In the course of these experiments, a glutamate-specific mutant amber suppressor, gluA-A73, was created. Finally, in the case of supF tyrosine tRNA, the discriminator is an important identity element with partial to complete loss of tyrosine specificity resulting from mutation at this position. It is clear from these experiments that it may not be possible to assign a specific role in tRNA identity to the discriminator. The identity of a tRNA in vivo is determined by competition among aminoacyl-tRNA synthetases, which is in turn modulated by the nucleotide substitution as well as the tRNA context.
J Mol Biol 1992 Dec 20
PMID:Synthetase competition and tRNA context determine the in vivo identify of tRNA discriminator mutants. 147 77

The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. Immunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. With prolonged exposure to dexamethasone (> 12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity to two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone.
Mol Endocrinol 1992 Dec
PMID:Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness. 149 90

In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-S-phase cultures, initiation bubbles were detected in several contiguous fragments from a previously defined 28-kb initiation locus. However, because of the low levels of bubblelike structures observed on gels, it has been suggested that these structures might represent artifacts, possibly unrelated to replication per se. In this study, we have achieved much more synchronous entry into S phase by using a novel inhibitor and have isolated replication intermediates by a new procedure that largely eliminates branch migration and shear. Under these conditions, we find that (i) the relative number of bubblelike structures detected in fragments from the initiation locus is markedly increased, (ii) bubbles are detected at multiple sites scattered throughout the region lying between the DHFR and 2BE2121 genes, and (iii) bubbles appear and disappear in this region with the kinetics expected of an early-firing origin. These data strengthen the proposal that in vivo, initiation can occur at any of a large number of sites scattered throughout a broad zone in the DHFR domain.
Mol Cell Biol 1992 Sep
PMID:Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid mimosine. 150 78

The polymerase chain reaction (PCR) was employed to detect Pneumocystis carinii in organs of infected rats. Using a pair of oligonucleotides designed to the dihydrofolate reductase (DHFR) gene of rat P. carinii, specific amplification of an expected 415 bp region of P. carinii DHFR DNA of this organism was achieved, while no amplification occurred with the human, Candida albicans, and Mycobacterium avium and tuberculosis DNAs. Using rat P. carinii isolated from in vitro cultures and infected lung homogenates, the minimum detection level by PCR on an ethidium bromide gel was about 200 organisms and by Southern analysis with radiolabelled DHFR probe the detection level improved to 20 organisms. This level of sensitivity is sufficient to detect P. carinii specific band on the gel in infected rat lung and other organs. This PCR technique is potentially useful for detecting P. carinii in bronchoalveolar lavage (BAL) fluids of AIDS patients and for quantifying the organisms in tissues and in in vitro cultures where a high background with conventional stains makes it harder to determine the number of organisms.
Mol Cell Probes 1992 Apr
PMID:Detection of Pneumocystis carinii in a rat model of infection by polymerase chain reaction. 151 43

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary.
Mol Cell Biol 1992 Mar
PMID:The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter. 154 88

We have used a modified cotransfection and selection strategy to create a series of mammalian cell lines that stably express high levels of intact glucocorticoid receptors. These cell lines were produced by subjecting Chinese hamster ovary (CHO) cells, which had been previously cotransfected with a glucocorticoid-responsive dihydrofolate reductase (DHFR) gene and the human glucocorticoid receptor gene, to growth in increasing concentrations of methotrexate (MTX). By linking the MTX selection process to glucocorticoid receptor function via the DHFR gene, stable cell lines resistant to a range of MTX concentrations (50 nM to 3 microM) were isolated that were strictly dependent upon glucocorticoids for growth. Quantitation of steroid binding capacity in MTX-resistant cells revealed a progressive increase in the number of glucocorticoid receptors as a function of increasing MTX concentration. This increase in receptor content was maximal at the highest level of MTX resistance examined (3 microM MTX) and represented a 25-fold elevation in glucocorticoid receptor number relative to CHO cells expressing only endogenous hamster receptor. The increases in steroid binding obtained after MTX selection were reflected by similar increases in the level of glucocorticoid receptor protein as determined by immunoblot analysis. Examination of glucocorticoid receptor structure by sucrose density gradient centrifugation revealed that oligomeric (9 S) steroid receptor complexes were formed at all levels of receptor expression. Subcellular localization of the glucocorticoid receptor protein by immunocytochemical staining revealed effective nuclear translocation of the overexpressed receptors in MTX-resistant cells. Functional transfection studies using a glucocorticoid-responsive reporter gene indicated that the additional glucocorticoid receptors in CHO cells were competent to activate transcription. To determine the molecular basis for the MTX-induced increases in functional glucocorticoid receptors, steady-state levels of glucocorticoid receptor mRNA were examined. MTX selection produced a 5- to 7-fold increase in transfected glucocorticoid receptor gene expression relative to untreated cells. MTX-resistant cells also expressed increased levels of a putative hamster glucocorticoid receptor mRNA species. Interestingly, the observed increases in receptor gene expression in these cells could not be accounted for by amplification of either the human or the hamster glucocorticoid receptor genes.
Mol Cell Endocrinol 1992 Feb
PMID:Methotrexate-induced overexpression of functional glucocorticoid receptors in Chinese hamster ovary cells. 154 9

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978-1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38-48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539-1546]. Transfection of DHFR- Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR+ phenotype with equal efficiency (i.e., greater than 2.5 x 10(-3), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR+ cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of greater than 8 x 10(-4). Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (888 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.
 ...
PMID:Construction of a dominant selectable marker using a novel dihydrofolate reductase. 155 67


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>