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)

Four cultured mammalian cell lines, differing in intrinsic resistance to methotrexate over a 70-fold range, have been compared with respect to several biochemical factors that might influence response to the drug. Cellular activity of the enzymes dihydrofolate reductase and thymidylate synthetase and the total levels of folate cofactors did not vary by more than a factor of 2 among the cell lines. All the cell types were able to transport extracellular methotrexate efficiently across the cell membrane, and at comparable rates. A kinetic study of highly purified dihydrofolate reductases from the four sources revealed small differences in the Km values for dihydrofolate and reduced nicotinamide adenine dinucleotide phosphate. A study was made of the inhibition of the four dihydrofolate reductases by methotrexate, and Ki values were obtained by fitting the Zone B equation of Goldstein (Goldstein, A., J. Gen. Physiol., 27: 529-580, 1944) to the resulting data. Values Ki determined by this method correlated with intrinsic resistance of the cell lines and showed a 25-fold range from the most sensitive to the most resistant line. It is concluded that the response of a cell to methotrexate is significantly influenced by the dissociation constant of its dihydrofolate reductase-methotrexate complex.
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PMID:Intrinsic resistance to methotrexate of cultured mammalian cells in relation to the inhibition kinetics of their dihydrololate reductases. 0 89

Six different R-factors conferring trimethoprim resistance had been isolated from a variety of sources. The trimethoprim-resistant dihydrofolate reductases (EC 1.5.1.3) from strains containing these R-factors were purified by ammonium sulphate precipitation and DEAE-cellulose ion-exchange chromatography. The enzymes showed no significant differences in molecular weight, pH profile, substrate profile, heat sensitivity, inhibition profile and Michaelis-Menten kinetics. There was, however, considerable variation in the specific activity of these enzymes in the same bacterial host. When two Escherichia coli trimethoprimsensitive dihydrofolate reductases were examined as controls, considerable differences between their properties and those of the enzymes mediated by R-factors were detected. The data suggest that one trimethoprim resistance gene could be spreading through the bacterial population, possibly situated on a transposon.
J Gen Microbiol 1978 Aug
PMID:R-factor mediated dihydrofolate reductases which confer trimethoprim resistance. 2 76

Three spontaneous fol regulatory mutants contain dihydrofolate reductase molecules which differ in physical properties from enzymes of their parent strains. The enzymes were purified over 100-fold by affinity chromatography and were shown to differ in vitro in thermolability and in affinity for trimethoprim, a competitive inhibitor of the enzyme. These results indicate that some fol regulatroy mutations occur in the structural gene for dihydrofolate reductase.
Mol Gen Genet 1977 Mar 07
PMID:Altered dihydrofolate reductase in fol regulatroy mutants of Escherichia coli K12. 32 67

DNA fragments of the R factor R388 which renders E. coli resistant to trimethoprim by inducing a trimethoprim resistant dihydrofolate reductase (Amyes and Smith, 1974) were inserted into plasmids and screened for the expression of the trimethoprim resistance gene. By means of a two step deletion procedure a 1770 bp EcoRI/BamH1 fragment was isolated which conferred drug resistance and which was found to induce the synthesis of the same dihydrofolate reductase as the parental R factor. Gene dosage experiments indicated that the induction was due to the presence of a dihydrofolate reductase structural gene on the 1770 bp fragment. The gene could be assigned to a segment which was less than 1200 bp long. The 1770 bp fragment and a recombinant plasmid consisting of pSF2124 and part of R388 were mapped with several restriction nucleases. The R factor induced enzyme was partially purified from a strain carrying a multicopy recombinant plasmid into which the 1770 bp fragment was inserted and which induced high levels of dihydrofolate reductase. The enzyme was found to be stable at 100 degrees. Some aspects of the synthesis of dihydrofolate reductase are discussed.
Mol Gen Genet 1978 Aug 04
PMID:Isolation of a small DNA fragment carrying the gene for a dihydrofolate reductase from a trimethoprim resistance factor. 36 38

Two clones from the Clarke-Carbon Escherichia coli colony bank were resistant to inhibition by trimethoprim, a potent inhibitor of dihydrofolate reductase. Both clones had elevated levels of dihydrofolate reductase. Furthermore, trimethoprim resistance and elevated enzyme levels were associated with ColE1 plasmids that carried DNA from the trkC ksgA pdxA region of the E. coli chromosome. Plasmid pLC1437a was shown by two criteria to carry the structural gene for dihydrofolate reductase: 1) A partial diploid containing plasmid pLC1437a produced a kinetically-recognizable dihydrofolate reductase that was not present in the parent haploid strain. 2) Plasmid pLC1437a coded for dihydrofolate reductase in vitro. A 1,000 base pair fragment of plasmid pLC1437a containing fol was used as a probe to measure fol mRNA in a mutant strain isolated by Sheldon and Brenner (Molec. gen. Genet. 147, 91-97, 1976). The mutation in this strain, which results in constitutively-high levels of dihydrofolate reductase and in the inability of the strain to grow at 42 degrees C, is cis dominant (Sheldon and Brenner, 1976). The results of kinetic hybridization and pulse-labeling experiments indicated that the regulatory mutant produced elevated levels of dihydrofolate reductase in response to an increased rate of synthesis of fol mRNA.
Mol Gen Genet 1979 Aug
PMID:Regulation of dihydrofolate reductase synthesis in Escherichia coli. 39 Mar 4

Trimethoprim inhibits dihydrofolate reductase. Mutations conferring trimethorpim-resistance on E coli K12 result in either an altered reductase with decreased affinity for the drug, or in 2-30 fold higher levels of the enzyme. Studies of the latter class of mutants indicate that dihydrofolate reductase is regulatdd by a diffusible molecule, and is probably under negative control. The regulatrory mutants, some of which are temperature-sensitive, act cis.
Mol Gen Genet 1976 Aug 10
PMID:Regulatory mutants of dihydrofolate reductase in Escherichia coli K12. 78 30

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

Making use of the polymerase chain reaction primed by oligonucleotides corresponding to regions conserved between members of the nucleoside monophosphate kinase family, we have isolated the yeast gene PAK3. Pak3p belongs to the subgroup of long-form adenylate kinase isozymes (deduced molecular mass 25.3 kDa) and exhibits highest sequence similarity to bovine AK3 rather than to the yeast isozyme, Aky2p. The gene is shown to be non-essential because haploid disruption mutants are viable, both in the presence and absence of a functional AKY2 allele. It maps on chromosome V upstream of RAD3. Its expression level is low when cells are grown on glucose or other fermentable carbon sources and about threefold higher on glycerol, but can be significantly induced by ethanol. A PAK3/mouse dihydrofolate reductase fusion construct expressed in yeast is targeted to mitochondria. Transformation with PAK3 on a multicopy plasmid complements neither adenylate kinase deficiency in an aky2-disrupted yeast strain nor in Escherichia coli cells conditionally defective in adenylate kinase.
Mol Gen Genet 1992 Jun
PMID:A new member of the adenylate kinase family in yeast: PAK3 is highly homologous to mammalian AK3 and is targeted to mitochondria. 162 94

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.
Mol Gen Genet 1991 Jun
PMID:Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate. 206 11

The type IIIb dihydrofolate reductase, a novel plasmid-encoded enzyme recently identified in Shigella sonnei, has been shown to have some similar biochemical properties to the type IIIa dihydrofolate reductase which was first identified in New Zealand in 1979. However, the type IIIb enzyme has a Ki for trimethoprim of 0.4 microM, and a pI of 5.35 (as compared to 19 nM and 6.1 for the type IIIa); both these results suggest that it is a different enzyme from the prototype type IIIa. The type IIIb dihydrofolate reductase was purified by methotrexate agarose affinity chromatography, yielding a pure protein as determined by HPLC. Automatic amino acid analysis of the purified enzyme showed it to be distinct from all other known plasmid-encoded dihydrofolate reductases and quite different from the type IIIa enzyme. The purified enzyme was examined by SDS-PAGE, which revealed that the type IIIb dihydrofolate reductase was a monomeric protein of Mr 17,200.
J Gen Microbiol 1990 Apr
PMID:N-terminal amino acid sequence of the novel type IIIb trimethoprim-resistant plasmid-encoded dihydrofolate reductase from Shigella sonnei. 220 77


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