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)

Dihydrofolate reductase from L1210 leukemia cells which are sensitive and resistant to methotrexate has the same physical and kinetic properties and immunoreactivity with a guinea pig antiserum raised to the enzyme purified from the methotrexate resistant strain. However, a chicken antiserum to dihydrofolate reductase from methotrexate sensitive L1210 cells has greater affinity for the homologous enzyme than for the enzyme from the MTX resistant cells indicating that there is some antigenic difference in these molecules.
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PMID:Immunologic heterogeneity of dihydrofolate reductase from methotrexate sensitive and resistant L1210 leukemia cells. 241 35

In beta-galactosidase of Escherichia coli residues 820-934 are similar to residues in dihydrofolate reductase of E. coli. Dihydrofolate reductase of E. coli and chicken are also similar and have identical tertiary structures. I used the similarity of the three-dimensional structure of prokaryotic and eukaryotic dihydrofolate reductases to align the chicken dihydrofolate reductase and the similar residues of beta-galactosidase. The positions of introns 1 and 5 of the chicken dihydrofolate reductase gene correspond exactly to the start and the end of the dihydrofolate reductase-like domain in the beta-galactosidase polypeptide chain. This equivalence of intron positions in a eukaryotic gene and domain structure in a prokaryotic protein was interpreted as evidence for a common origin of both genes.
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PMID:The border residues of the dihydrofolate reductase domain in Escherichia coli beta-galactosidase correspond to the positions of introns 1 and 5 of dihydrofolate reductase of chicken. 250 33

The beta subunit of the human fibronectin receptor (FNRB) is a transmembrane protein belonging to the VLA (very late antigens of activation) family. Using pGEM-32, a 2.5-kb partial cDNA clone corresponding to the 3' portion of the human FNRB locus, multiple restriction fragment length polymorphisms (RFLPs) were revealed on DNAs from unrelated Caucasians. RFLPs detected by five enzymes, BanII, HinfI, KpnI, BglII, and SacI, are of the simple two-allele form, and pairwise linkage analyses of these RFLPs with numerous known DNA markers from the chromosome-10 pericentromeric region not only confirmed the chromosome-10 assignment of the functional FNRB gene but also supported its localization at p11.2 suggested by in situ hybridization. An infrequent MspI RFLP was detected by pB/R2, a 4.6-kb genomic clone from the FNRB locus. Another type of DNA polymorphism was also revealed by the cDNA clone and it was visualized on the Southern blot analyses as the presence or absence of an extra band (or a set of extra bands). It seems to stem from a stretch of DNA sequence present in some individuals at one single locus but absent in others, and is of non-chromosome-10 origin based on linkage analyses with known chromosome 10 markers. This "presence/absence" type of polymorphism could be revealed by all of the 25 restriction enzymes tested and is similar in nature to that previously reported with one of the human dihydrofolate reductase pseudogenes, DHFRP1. Dissection of the pGEM-32 clone demonstrated that the region revealing the non-chromosome-10 sequences is within a fragment about 1.7 kb in length extending from about 600 nucleotides preceding the stop codon down to the end of the cloned FNRB 3' untranslated region. Due to its high polymorphism information content (PIC) value (0.71 for haplotypes of BanII, HinfI, and KpnI RFLPs) and proximity to the centromere. FNRB will prove to be a highly useful marker for genetic linkage studies of multiple endocrine neoplasia type 2A (MEN2A) as well as for chromosome-10 linkage studies in general.
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PMID:The beta subunit locus of the human fibronectin receptor: DNA restriction fragment length polymorphism and linkage mapping studies. 257 37

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.
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PMID:Characterization of Candida albicans dihydrofolate reductase. 264 98

Dihydrofolate reductase with a molecular weight of 22,000 has been purified by salt precipitation and methotrexate-Sepharose affinity chromatography from mouse livers having a mean weight of 2.4 g each. When the same purification procedure was followed using livers with a mean weight of 1.4 g or less, a protein with a molecular weight of 27,500 co-purified with dihydrofolate reductase. This 27.5 kDa species was recovered with dihydrofolate reductase following a second passage through the affinity column and it reacted by Western immunoblotting with a monospecific polyclonal antiserum raised to the purified 22 kDa enzyme. The two proteins could not be separated in a native state to compare their functional activity, but the 27.5 kDa protein appeared to have catalytic reductase activity when assayed directly on the polyacrylamide gel following non-denaturing electrophoresis. The catalytic activity of the mixture of the purified proteins was stoichiometrically inhibited by a molar equivalent of methotrexate.
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PMID:Age-dependent expression of a novel protein in mouse liver immunologically and functionally homologous with dihydrofolate reductase. 280 23

Resistance to trimethoprim (Tp) is mediated by a plasmid-encoded gene in staphylococci. The gene is responsible for high-level resistance (MIC, greater than 1,000 micrograms/ml) in both its native host and when cloned on high-copy-number vectors in Escherichia coli. Analysis of subclones of the staphylococcal Tp gene on E. coli expression vectors and estimation of the size of full and truncated proteins produced in E. coli minicells generated an approximate size limit of 505 base pairs for the gene and 18,500 daltons for the gene product. Crude extracts of E. coli containing the cloned gene had dihydrofolate reductase (DHFR) specific activity that was more than 100 times greater than that of control cells and more than 1,000 times more resistant to trimethoprim inhibition. The amount of trimethoprim required for a 50% reduction in the specific activity of staphylococcal DHFR differed from those of cells containing DHFR types I, II, or III, enzymes mediating Tp in members of the family Enterobacteriaceae. In addition, the size of the monomeric staphylococcal DHFR protein was larger than that of any of the gram-negative DHFRs both compared with published sequence data and as observed by direct comparison on polyacrylamide gels. Finally, there was no homology between a DNA fragment containing the cloned staphylococcal gene and DNA encoding any of the gram-negative DHFRs. Thus, the staphylococcal Tp gene codes for a single protein with DHFR activity that appears to be unrelated to DHFR genes that mediate Tp in members of the Enterobacteriaceae.
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PMID:Characterization of a staphylococcal trimethoprim resistance gene and its product. 282 86

The ADP/ATP carrier of yeast (309 amino acids) is an abundant transmembrane protein of the mitochondrial inner membrane whose import involves well-defined steps (Pfanner, N., and Neupert, W. (1987) J. Biol. Chem. 262, 7528-7536). Analysis of the in vitro import of gene fusion products containing ADP/ATP carrier (AAC) sequences at the amino terminus and mouse dihydrofolate reductase (DHFR) at the carboxyl terminus indicates that the first 72 amino acids of the soluble carrier protein, a hydrophilic region of the protein, are not by themselves sufficient for initial binding to the AAC receptor on the mitochondrial surface. However, an AAC-DHFR gene fusion containing the first 111 residues of the ADP/ATP carrier protein exhibited binding to mitochondria at low temperature (2 degrees C) and internalization at 25 degrees C to a mitochondrial space protected from proteinase K in the same manner as the wild-type ADP/ATP carrier protein. The AAC-DHFR protein, in contrast to the wild-type AAC protein imported into mitochondria under optimal conditions, remained extractable at alkaline pH and appeared to be blocked at an intermediate step in the AAC import pathway. Based on its extraction properties, this AAC-DHFR hybrid is proposed to be associated with a proteinaceous component of the import apparatus within mitochondria. These data indicate that the import determinants for the AAC protein are not located at its extreme amino terminus and that protein determinants distal to the first 111 residues of the carrier may be necessary to move the protein beyond the alkali-extractable step in the biogenesis of a functional AAC protein.
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PMID:Mitochondrial import of the ADP/ATP carrier protein in Saccharomyces cerevisiae. Sequences required for receptor binding and membrane translocation. 283 88

We studied 10 trimethoprim-resistant (Tmpr) Haemophilus influenzae isolates for which agar dilution MICs were 10 to greater than 200 micrograms/ml. Trimethoprim resistance was transferred from two Tmpr H. influenzae isolates to a Tmps strain by conjugation or transformation. Wild-type Tmpr strains and Tmpr transcipients did not contain detectable plasmid DNA. The trimethoprim resistance gene was cloned into a cosmid vector, and recombinant plasmids were transduced into Escherichia coli. A 0.50-kilobase intragenic probe derived from a 12.9-kilobase fragment which encoded trimethoprim resistance hybridized with whole-cell DNA from Tmps and Tmpr strains. Southern blot analysis of restricted DNA from isogenic Tmps and Tmpr H. influenzae indicated that acquisition of trimethoprim resistance involved a rearrangement or change in nucleotide sequence. Hybridization was not seen with DNA derived from Tmpr E. coli containing dihydrofolate reductase I, II, and III genes or with Tmpr Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas cepacia. Southern hybridization with 12 multiply resistant encapsulated H. influenzae strains confirmed that the trimethoprim resistance gene was chromosomally mediated. Dihydrofolate reductase activity was significantly greater in cell sonicate supernatants of Tmpr strains in comparison with isogenic Tmps recipients. Differences were not found in the trimethoprim inhibition profile of dihydrofolate reductase activity in Tmps and Tmpr strains. We conclude that the mechanism of trimethoprim resistance in H. influenzae is overproduction of chromosomally located dihydrofolate reductase.
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PMID:Molecular cloning and mechanism of trimethoprim resistance in Haemophilus influenzae. 283 38

The effects of productive adenovirus infection on host gene expression were studied by using a line of methotrexate-resistant HeLa cells with amplified dihydrofolate reductase (DHFR) genes. We have previously reported that synthesis of DHFR is induced threefold early in infection and is shut off late in infection (Yoder et al., Mol. Cell. Biol. 3:819-828, 1983). These changes in DHFR protein synthesis are accompanied by changes in both the steady-state cytoplasmic levels of DHFR mRNA and in the rate of appearance of DHFR mRNA in the cytoplasm. In this report, we examined the mechanism of nuclear control of DHFR mRNA levels. Transcription of DHFR-specific sequences continued at a constant rate throughout infection, representing 0.015% of the total transcriptional activity. In contrast, nuclear steady-state levels of DHFR sequences changed in correspondence to the changing rate of appearance of DHFR mRNA in the cytoplasm. That is, nuclear levels of DHFR-specific sequences rose 2.5-fold early in infection and declined to a level below that found in uninfected cells late in infection. Thus, the relative nuclear stability of DHFR sequences changed throughout the course of infection such that during the time of induction, DHFR sequences were preferentially stabilized. This stabilization was transient, however, and was no longer observed by the time of shutoff. These data indicate that posttranscriptional nuclear events are important in the regulation of DHFR gene expression by adenovirus.
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PMID:Posttranscriptional control of DHFR gene expression during adenovirus 2 infection. 298 22

Quantitative S1 nuclease mapping studies were performed with uniformly labeled RNA probes, containing contiguous dihydrofolate reductase exon and intron sequences, and total RNA isolated from KB7B cells exposed to 5-fluorouracil for 5 days. Dihydrofolate reductase RNA containing both exon 1 and intron I, or exon 5 and a portion of intron V, increased up to 5-fold in cells grown in the presence of 2.0 to 3.0 microM 5-fluorouracil. Dihydrofolate reductase RNA containing exon 1 or exon 5, but lacking intron I or intron V, respectively, increased 2-fold in cells grown in the presence of 0.65 to 3.0 microM 5-fluorouracil. Primer extension analysis and S1 mapping studies revealed two major transcriptional start sites at positions -72 and -69 and minor start sites upstream from position -183, for dihydrofolate reductase RNA isolated from methotrexate-resistant KB7B cells. The results of these studies demonstrate that 5-fluorouracil alters the metabolism of dihydrofolate reductase precursor mRNA and/or processing intermediates.
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PMID:5-Fluorouracil augmentation of dihydrofolate reductase RNA containing contiguous exon and intron sequences in KB7B cells. 303 31


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