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)

Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors. Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1. The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps. Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity. Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity. Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100. The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.
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PMID:Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities. 217 3

Triplex-forming oligonucleotides (TFOs) have been shown to bind to target DNA sequences in several human gene promoters such as the c-myc oncogene, the epidermal growth factor receptor, and the dihydrofolate reductase genes. TFOs have been shown to inhibit transcription in vitro and gene expression in cell culture of the c-myc and other genes. The HER-2/neu oncogene, which is overexpressed in breast cancer and other human malignancies, contains a purine-rich sequence in its promoter, which is favorable for purine:purine:pyrimidine (R:R:Y) triplex formation. Although its function in the HER-2/neu promoter is unknown, this purine-rich site is homologous to a protein-binding sequence in the promoter of the epidermal growth factor receptor that is necessary for efficient transcription of this gene. We have shown that this sequence is a site for nuclear protein binding by incubation with a crude nuclear extract. We describe the formation of an interstrand triplex using a purine-rich oligonucleotide antiparallel to this purine-rich target sequence of the HER-2/neu promoter. Triplex formation by the oligonucleotide prevents protein binding to the target site in the HER-2/neu promoter in vitro. We have shown that this oligonucleotide is a potent and specific inhibitor of HER-2/neu transcription in an in vitro assay. The triplex target site contains a single pyrimidine base that does not conform to the R:R:Y triplex motif. In an attempt to abrogate the potentially destabilizing effects of this pyrimidine base on triplex formation, we have substituted an abasic linker for the pyrimidine residue in the triplex forming oligonucleotide. Triplex formation with the modified oligonucleotide appears to occur with approximately equivalent binding affinity. Triplex formation in the HER-2/neu oncogene promoter prevents transcription in vitro and may represent a future modality for specific inhibition of this gene in vivo.
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PMID:Triplex formation inhibits HER-2/neu transcription in vitro. 790 Dec 37

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg83b27/vgBG heterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.
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PMID:The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis. 823 10

The vestigial (vg) gene of Drosophila melanogaster encodes a nuclear protein which plays a key role in wing formation but is also involved in other developmental processes. We have previously shown that depletion of the dTMP pool by aminopterin, an inhibitor of the enzyme dihydrofolate reductase, or by fluorodeoxyuridine, an inhibitor of thymidylate synthetase, induces nicks in the wings of wild-type flies and a strong vg phenotype in vgBG/+ flies and also in individuals heterozygous for a deficiency of the vg locus (vgB/+). Furthermore, specific alterations of the vg locus, caused by intronic insertions, are associated with resistance to these drugs. In this paper, we show that: (1) depletion of the dTMP pool by aminopterin leads to a decrease in the amount of vg transcripts; (2) insertion of the retrotransposon 412 in the vgBG mutant, which is resistant to aminopterin, leads to the formation of a truncated transcript that is prematurely terminated in the long terminal repeat of this transposable element; and (3) aminopterin also affects the level of this truncated transcript. These results indicate that alterations of the wing by inhibitors of dTMP synthesis are caused by an effect of these drugs on levels of vg transcripts; the resistance to such agents observed for the vgBG strain is not due to a qualitatively different effect of this drug on the vg transcript but, rather, is related to the expression of a modified Vg protein encoded by a truncated transcript. These results are compatible with a role for vestigial in modulating cell proliferation.
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PMID:Vestigial gene expression in Drosophila melanogaster is modulated by the dTMP pool. 862 52

RING3 is a novel protein kinase linked to human leukaemia. Its Drosophila homologue female sterile homeotic is a developmental regulator that interacts genetically with trithorax, a human homologue of which is also associated with leukaemia. The RING3 structure contains two mutually related bromodomains that probably assist in the remodelling of chromatin and thereby affect transcription. Consistent with this hypothesis, a RING3-like protein has been identified in the mouse Mediator complex, where it is associated with transcription factors. We show that, whilst RING3 is constitutively localised to the nucleus of exponentially growing HeLa cells, it is delocalised throughout serum-starved fibroblasts. We use immunostaining and confocal microscopy to demonstrate that RING3 translocates to the fibroblast nucleus upon serum stimulation. After translocation, RING3 participates in nuclear protein complexes that include E2F proteins; it transactivates the promoters of several important mammalian cell cycle genes that are dependent on E2F, including dihydrofolate reductase, cyclin D1, cyclin A and cyclin E. We use site-directed mutagenesis of a putative nuclear localisation motif to show that the activation-induced nuclear localisation and consequent transcriptional activity of RING3 depends on a monopartite, classical nuclear localisation sequence. These observations refine and extend the mechanism by which RING3 contributes to E2F-regulated cell cycle progression. Deregulation of this mechanism may be leukaemogenic.
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PMID:Activation-induced nuclear translocation of RING3. 1093 46

Conditional mutants are a vital tool for analysis of gene function. The use of temperature-sensitive mutants in Schizosaccharomyces pombe has significantly promoted understanding of many cellular processes. A portable heat-inducible amino-terminal degron (N-degron) for conditional degradation of a gene product has been previously described in Saccharomyces cerevisiae. This paper describes the adaptation of the N-degron method to create temperature-sensitive (ts) mutants in S. pombe. A ts derivative of the mouse dihydrofolate reductase with an amino-terminal arginine (Arg-DHFR(ts)) previously described in S. cerevisiae was fused to the N-terminus of Bir1p, a nuclear protein involved in mitotic chromosome segregation in S. pombe. This fusion allele, referred to as bir1-td, conferred a chromosome segregation defect at 36 degrees C, as with previously described alleles of bir1. Deletion of the S. pombe E3 ubiquitin ligase (N-recognin), Ubr11p, reversed the temperature-dependent lethality of bir1-td, providing evidence for N-end rule mediated destruction of Bir1p. The methods we describe should therefore facilitate analysis of essential genes in fission yeast for which conditionally lethal mutants are unavailable.
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PMID:The N-degron approach to create temperature-sensitive mutants in Schizosaccharomyces pombe. 1515 87