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 degree of methylation at c-myc proto-oncogene was found to change during aging process of mice by the use of methylation-sensitive restriction enzymes. The spleen DNA showed hypomethylation as mice aged, while hypermethylation was observed in the liver DNA. The brain DNA on the contrary revealed no appreciable difference between young and old mice. When the DNAs were examined at actin and dihydrofolate reductase (DHFR), no significant change was observed. It suggests that an age-related change of oncogene structure may be one of the factors which are related to an age-associated increase of cancer incidence rate.
Biochem Biophys Res Commun 1986 Sep 30
PMID:Methylation of the c-myc gene changes during aging process of mice. 302 Nov 54

Site-directed mutagenesis was used to generate mutants of recombinant mouse dihydrofolate reductase to test the role of some amino acids in the binding of two inhibitors, methotrexate and trimethoprim. Eleven mutations changing eight amino acids at positions all involved in hydrogen bonding or hydrophobic interactions with dihydrofolate or one of the two inhibitors were tested. Nine mutants were obtained by site-directed mutagenesis and two were spontaneous mutants previously obtained by in vivo selection (Grange, T., Kunst, F., Thillet, J., Ribadeau-Dumas, B., Mousseron, S., Hung, A., Jami, J., and Pictet, R. (1984) Nucleic Acids Res. 12, 3585-3601). The choice of the mutated positions was based on the knowledge of the active site of chicken dihydrofolate reductase established by x-ray crystallographic studies since the sequences of all known eucaryotic dihydrofolate reductases are greatly conserved. Enzymes were produced in great amounts and purified using a plasmid expressing the mouse cDNA into a dihydrofolate reductase-deficient Escherichia coli strain. The functional properties of recombinant mouse dihydrofolate reductase purified from bacterial extracts were identical to those of dihydrofolate reductase isolated from eucaryotic cells. The Km(NADPH) values for all the mutants except one (Leu-22----Arg) were only slightly modified, suggesting that the mutations had only minor effects on the ternary conformation of the enzyme. In contrast, all Km(H2folate) values were increased, since the mutations were located in the dihydrofolate binding site. The catalytic activity was also modified for five mutants with, respectively, a 6-, 10-, 36-, and 60-fold decrease of Vmax for Phe-31----Arg, Ile-7----Ser, Trp 24----Arg and Leu-22----Arg mutants and a 2-fold increase for Val-115----Pro. All the mutations affected the binding of methotrexate and six, the binding of trimethoprim: Ile-7----Ser, Leu-22----Arg, Trp-24----Arg, Phe-31----Arg, Gln-35----Pro and Phe-34----Leu. The relative variation of Ki for methotrexate and trimethoprim were not comparable from one mutant to the next, reflecting the different binding modes of the two inhibitors. The mutations which yielded the greatest increases in Ki are those which involved amino acids making hydrophobic contacts with the inhibitor.
J Biol Chem 1988 Sep 05
PMID:Site-directed mutagenesis of mouse dihydrofolate reductase. Mutants with increased resistance to methotrexate and trimethoprim. 304 18

Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [3H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3+ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3- lymphocytes. Stimulation of CD3+ and CD3- lymphocyte subpopulations induced by env-gag peptides was specifically inhibited by goat anti-env-gag polyclonal antibodies, demonstrating the specificity of the reaction. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection.
Proc Natl Acad Sci U S A 1988 Sep
PMID:Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes. 304 18

The promoter of the human c-K-ras gene has been characterized by deletion mutagenesis in concert with stable and transient expression gene transfer experiments. The transcription initiation sites were determined by S1 mapping and RNase A protection experiments. The c-K-ras promoter region is rich in G + C, lacks TATA and CCAAT boxes and contains sequence similarities with other house-keeping genes such as the dihydrofolate reductase (DHFR) and the epidermal growth factor (EGF) receptor genes. The promoter of the c-K-ras gene consists of multiple elements and initiation of transcription occurs at multiple sites. A 54 bp DNA fragment immediately upstream from the 5' end untranslated exon controls the position of many of the transcription initiation sites and direct sufficient transcription for transformation of NIH3T3 cells. However, these sequences can be replaced by other upstream sequences which are required for optimal gene expression. In addition, sequences overlapping with the 5' end untranslated exon and therefore downstream from the major transcription initiation sites are important (although not sufficient) for transcription because their deletion greatly impairs the promoter activity of the upstream elements.
Oncogene Res 1988 Sep
PMID:Characterization of the human c-K-ras gene promoter. 306 87

The poly-gamma-glutamyl derivatives of n10-propargyl-5,8-dideazafolic acid (PDDF) with a chain length of up to five glutamate residues were synthesized from N10-propargyl-5,8-dideazapteroic acid by the solid-phase procedure. These compounds were evaluated for their antifolate activity using folate-requiring microorganisms and intact and permeabilized L1210 cells and as inhibitors of dihydrofolate reductase and thymidylate synthase derived from L. casei. The polyglutamylated derivatives of PDDF (1) were more active than the parent compound in inhibiting the growth of L. casei, thymidylate synthesis in permeabilized L1210 cells, and L. casei thymidylate synthase. Two analogues of 5,8-dideazafolic acid (2 and 3), one with a 2-butyne and another with a cyclopropylmethyl substituent at N10, were also synthesized and evaluated for their antifolate activities using the above-mentioned test systems. They were considerably less active than PDDF or its polyglutamylated derivatives. N10-Propargyl-5,8-dideazapteroyl tri-, tetra-, and pentaglutamates were equipotent with 5-fluorodeoxyuridylate as inhibitors of thymidylate synthesis in permeabilized L1210 cells. The polyglutamyl metabolites of PDDF were shown to be the most potent antifolate inhibitors of L. casei and L1210 thymidylate synthases yet described.
J Med Chem 1986 Sep
PMID:Folate analogues. 26. Syntheses and antifolate activity of 10-substituted derivatives of 5,8-dideazafolic acid and of the poly-gamma-glutamyl metabolites of N10-propargyl-5,8-dideazafolic acid (PDDF). 309 32

The synthesis of the 5,10-methylene analogue of 5,6,7,8-tetrahydro-8,10-dideazaminopterin, a potential dual inhibitor of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzymes, is described. The dimethyl ester of 10-carboxy-4-amino-4-deoxy-8,10-dideazapteroic acid was converted to the tetrahydro derivative by hydrogenation. Thermally induced cyclization of the 10-carbomethoxy and the 5-NH groups afforded the 5,10-carbonyl analogue. Reduction of the lactam with borane readily yielded the key 5,10-methylene-4-amino-4-deoxy-8,10-dideazatetrahydropteroic acid methyl ester. Saponification of the benzoate ester and coupling with L-glutamate concluded the synthesis. The title compound was a modest inhibitor of growth in folate-dependent bacteria. Streptococcus faecium and Lactobacillus casei, but inhibition of DHFR or TS derived from L. casei was poor. The compound was also a weak inhibitor of DHFR derived from L1210 murine leukemia and was a weak inhibitor of L1210 growth in culture.
J Med Chem 1986 Sep
PMID:Synthesis and antifolate properties of 5,10-methylenetetrahydro-8,10-dideazaminopterin. 309 34

Agents that inhibit DNA synthesis increase the frequency of methotrexate resistance and gene amplification in cultured mammalian cells. Chinese hamster ovary cells blocked with hydroxyurea rereplicated dihydrofolate reductase gene sequences within a single cell cycle upon release from the block (Mariani, B.D., and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). Perturbation of DNA synthesis was postulated to result in misfiring of replicon initiation, subsequent over-replication of DNA sequences, and amplification of specific genes. To test this hypothesis, we have exposed Chinese hamster ovary cells pulsed with bromodeoxyuridine to three agents that inhibit DNA synthesis and enhance gene amplification: UV irradiation, hydroxyurea, and aphidicolin. After release from the block, the progression of cells throughout the cell cycle was analyzed by flow cytometry through simultaneous measurement of total cellular DNA content and bromodeoxyuridine-labeled DNA. Although the cell cycle effects varied depending on the agent used for the block, in all cases a subset of cells that were in S phase at the time of the block exhibited DNA histograms with greater than 4C DNA content at various times after release and prior to cell division. Cells with the excess DNA were approximately 10-fold more resistant to methotrexate compared to treated cells with normal DNA content or untreated cells. Therefore, cells in S phase at the time of the block produce excess DNA per cell prior to division, and this over-replicated DNA may be relevant to gene amplification and drug resistance.
J Biol Chem 1987 Sep 05
PMID:Over-replication of DNA in S phase Chinese hamster ovary cells after DNA synthesis inhibition. 311 56

The transfer of the adenovirus 5 E1A gene into brain cells from rat embryos led to the establishment of phenotypically untransformed clonal glial cell lines. Some lines displayed properties of oligodendrocyte-astrocyte progenitors. Their differentiation involved several steps that were dependent on culture conditions and cell interactions. Subsequently, a few lines were cotransfected with a vector carrying a modified dihydrofolate reductase gene and with the Escherichia coli lacZ gene. After selection for resistance to methotrexate, cell lines were derived that stably expressed the lacZ gene. These cells were individually detectable by histochemical staining for beta-galactosidase activity, even in the presence of other cells. These results suggest that this type of cell line could be of interest for further in vitro, and possibly transplant, studies of the differentiation and interactions of glial cells.
J Neurosci Res 1988 Sep
PMID:Immortalization of bipotential glial progenitors and generation of permanent "blue" cell lines. 314 80

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E. coli. The B. subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RBS, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low.
Gene 1988 Sep 15
PMID:Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system. 314 23

DNA probes for trimethoprim-resistant dihydrofolate reductases types IV and V were constructed and characterized. The type IV probe consisted of a 1.7 kb ClaI fragment of recombinant plasmid pUK1148, while the type V probe was a 0.5 kb HincII fragment of plasmid pLK09. Each probe was biotinylated and tested in non-isotopic hybridization experiments with control plasmids carrying genes for known dihydrofolate reductase types. Both probes were highly specific and will therefore be useful in epidemiological studies for monitoring the spread of these unusual plasmid-encoded enzymes.
J Antimicrob Chemother 1988 Sep
PMID:Biotinylated DNA probes for trimethoprim-resistant dihydrofolate reductases types IV and V. 318 27


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