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

The hydroxyurea-resistant Chinese hamster cell line 600H has been shown to have greatly elevated quantities of ribonucleotide reductase. This increase in enzyme activity is due to an increased level of both the M1 and M2 subunit activities. The M1 subunit has been purified from the 600H cell line and shown to consist of a series of six protein spots with apparent molecular weights of 88,000 daltons, but with varying isoelectric points in the range of pH 6.5-7.0. Western blot analyses with antisera against the M1 and M2 proteins indicated that both subunit proteins are present in elevated quantities in the 600H cell line when compared to the wild-type V79 cell line. Southern blot analyses with genomic DNA from the series of stepwise-selected hydroxyurea-resistant cell lines leading to 600H showed that, in latter steps of selection, genomic sequences homologous to a mouse M1 cDNA have undergone a fivefold amplification. This was accompanied by a four- to eightfold increase in the single M1 homologous mRNA.
Somat Cell Mol Genet 1987 May
PMID:Gene for M1 subunit of ribonucleotide reductase is amplified in hydroxyurea-resistant hamster cells. 329 47

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.
Mol Cell Biol 1987 Aug
PMID:Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability. 331 4

A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.
Mol Cell Biol 1987 Dec
PMID:Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells. 343 88

Hydroxyurea was used as a selective agent in culture, to isolate by a stepwise procedure, a unique mouse L cell line called LHF which exhibited a stable resistance to high concentrations of drug (5 mM). LHF cells contained an elevation in ribonucleotide reductase activity which depended upon whether cells were previously cultured in the presence or absence of hydroxyurea. M1 immunoprecipitation and M2 titration experiments indicated that both ribonucleotide reductase subunits were elevated in drug-resistant cells. Interestingly, a very large drug-dependent change in the M2 activity (about a 100-fold) was observed. Studies on enzyme activity with cycloheximide and actinomycin D indicated that the hydroxyurea-dependent increase in activity required de novo protein synthesis and transcriptional activity. These results are different from other ribonucleotide reductase overproducing cell lines previously described, and indicate that hydroxyurea modulates enzyme activity by an interesting mechanism.
Somat Cell Mol Genet 1986 Mar
PMID:Characterization of a mouse cell line selected for hydroxyurea resistance by a stepwise procedure: drug-dependent overproduction of ribonucleotide reductase activity. 351 84

Five hamster, mouse, and rat cell lines resistant to the cytotoxic effects of hydroxyurea have been characterized. All cell lines contained increased ribonucleotide reductase activity, elevated levels of the M2 component of ribonucleotide reductase as judged by electron paramagnetic resonance spectroscopy, and increased copies of M2 mRNA as determined by Northern blot analysis. Two species of M2 mRNA were detected in rodent cell lines, a high-molecular-weight species of approximately 3.4 kb in hamster and rat cells and about 2.1 kb in mouse cells. The low molecular-weight M2 mRNA was about 1.6 kb in all rodent lines. Northern blot analysis showed that the mRNA for the other component of ribonucleotide reductase, M1, was not markedly elevated in the drug-resistant cells and existed as a single 3.1-kb species. Four of the five resistant lines contained an M2 gene amplification as determined by Southern blot analysis, providing direct evidence to support earlier suggestions that hydroxyurea resistance is often accompanied by amplification of a ribonucleotide reductase gene. An increase in gene dosage was detected even in cells exhibiting only modest drug-resistance properties. No evidence for amplification of the M1 gene of ribonucleotide reductase was found. In keeping with these observations with drug-resistant rodent lines, a human (HeLa) cell line resistant to hydroxyurea was also found to contain increased levels of two M2 mRNA species (about 3.4 and 1.6 kb) and exhibited M2 gene amplification. One hamster cell line resembled the other resistant rodent lines in cellular characteristics but did not show amplification of either the M1 or M2 gene, providing an example of a drug-resistant mechanism in which an elevation of M2 mRNA has occurred without a concomitant increase in M2 gene copy number.
Somat Cell Mol Genet 1987 Mar
PMID:Altered expression of ribonucleotide reductase and role of M2 gene amplification in hydroxyurea-resistant hamster, mouse, rat, and human cell lines. 355 Nov 13

This paper presents a dual-recognition model of the T-cell receptor that has been constructed to account for the phenomenon of MHC restriction as well as the paradoxical ability of T-cells to be both multispecific and precisely specific at the same time. In our model the combining sites for antigen and MHC are not independent as in classical dual-recognition models, but interact with each other by an allosteric mechanism. We envision a flexible receptor with combining sites for antigen and MHC that are capable of existing in a multitude of distinct complementarity states. MHC and antigen molecules act as allosteric effectors such that one ligand perturbs the conformation and therefore the specificity of the site for the other ligand. An essential feature of the model is that different MHC determinants induce different conformations at the anti-antigen site. In this way the receptor acquires multiple specificities. Within a particular complementarity state, precise recognition results from the requirement that antigen and MHC exhibit positive cooperativity in their binding to the T-cell receptor. Positive cooperativity is also the basis for MHC restriction. Reaction mechanisms are presented which describe the requirement that antigen and MHC both induce conformational changes in order to generate high-affinity binding to either ligand. As a precedent for the multistate allosteric receptor model, we discuss the properties of allosteric enzymes, especially ribonucleotide reductase, whose properties are analogous to those we have postulated for the T-cell receptor. Also discussed is the possibility that molecules such as Ly2, L3T4 and the Mls antigen, which have been found to play a role in antigen recognition, function as affinity-enhancing allosteric effectors that interact with the constant portion of the T-cell receptor.
Mol Immunol 1984 Nov
PMID:Hypothesis: the MHC-restricted T-cell receptor as a structure with two multistate allosteric combining sites. 608 44

The metabolic consequences of deoxyuridine treatment in four cultured human lymphoblast lines (CCRF-CEM, RPMI-8402, JM, and BALM) were studied by cell growth experiments, flow cytometry, and measurement of 2'-deoxyribonucleoside triphosphate (dNTP) levels. DNA perturbations occurred in all lymphoblast lines, but there was no significant impairment of RNA synthesis. The DNA perturbations in CCRF-CEM, RPMI-8402, and BALM cells reflected inhibition of DNA synthesis, and the associated dNTP changes were consistent with ribonucleotide reductase inhibition or, specifically in BALM cells, with DNA alpha-polymerase inhibition. JM cells treated with an intermediate concentration of deoxyuridine developed a block at the G1/S boundary which was deoxyuridine concentration-dependent, but not specific for deoxyuridine (it was also seen with thymidine treatment) and not related to DNA synthesis inhibition. There were no idiosyncratic dNTP effects accompanying the G1/S boundary block, and the responsible metabolic mechanism remains to be determined.
Mol Pharmacol 1982 Jul
PMID:The metabolic basis of deoxyuridine cytotoxicity. Studies of cultured human lymphoblasts. 618 85

Hydroxyurea inhibited the growth and DNA synthesis of Toxoplasma gondii growing in human fibroblast cells. A concentration of 18 micrograms/ml totally suppressed plaque formation. The synthesis of T. gondii RNA was not acutely inhibited. The parasite was equally sensitive to hydroxyurea when grown in wild type or hydroxyurea resistant host cells. With the aid of chemical mutagenesis, we isolated a stable hydroxyurea resistant mutant of T. gondii. This mutant showed no increased ability to incorporate [3H]uracil into its pyrimidine deoxynucleotide pool. Hydroxyurea depressed the [3H]uracil labeling of the pyrimidine deoxynucleotide pool in the wild type parasite but not in the mutant, suggesting that the mutant ribonucleotide reductase was resistant to the inhibitory effect of the drug.
Mol Biochem Parasitol 1982 Sep
PMID:Hydroxyurea inhibition of growth and DNA synthesis in Toxoplasma gondii: characterization of a resistant mutant. 618 67

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.
Mol Cell Biol 1983 Jun
PMID:Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity. 630 22

Human T leukemic lymphocytes are highly susceptible to growth inhibition by dAdo. We have investigated this phenomenon using analytical DNA flow cytometry. By using (a) bromodeoxyuridine quenching of Hoechst 33342 fluorescence and (b) the drug ICRF-159, a selective G2 - M-blocking agent, we show that dAdo causes a G1 block in cultured T leukemic cells and that cells in the S phase exposed to dAdo are able to complete that S phase, pass through G2 + M, and return to the G1 phase. Centrifugal elutriation was used to enrich cells for various phases of the cell cycles. dAdo caused elevation of the dATP pool to a similar extent in G1, S, and G2 - M-enriched cell fractions, but did not cause a fall in the dCTP pool. These findings indicate that dAdo induces a G1 block via elevation of dATP pools, apparently independently of inhibition of ribonucleotide reductase.
Mol Pharmacol 1984 Sep
PMID:Analytical DNA flow cytometric analysis of deoxyadenosine toxicity in cultured human leukemic lymphoblasts. 633 78


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