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)

We described rapid methods to detect Y-specific repeated DNA sequences in cytological preparations using in situ hybridization. A human Y chromosome specific DNA probe with an insert equivalent to that in pHY2.1 was labelled with [alpha-32P]dCTP or photobiotin, and hybridized to chromosome preparations. Signals were visualized specifically on Y chromosomes after 1 day's autoradiography or a couple of hours treatment with streptavidin alkaline phosphatase/BCIP/NBT. These methods are useful for molecular confirmation of Y-autosomal translocations.
Cell Mol Biol 1989
PMID:Rapid methods to visualize Y-specific repeated DNA sequences in cytological preparations. 265 88

Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step. Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions. The latter pathway should be sensitive to deoxyribonucleoside triphosphate (dNTP) pool fluctuations. Since dNTP pools are regulated through allosteric control of ribonucleotide reductase, the control of this enzyme should be a determinant of BrdUrd mutagenesis--if mutagenesis results largely from incorporation errors. Since T4 phage-encoded ribonucleotide reductase is insensitive to feedback inhibition, we established conditions under which phage DNA replication is dependent upon ribonucleotide reductase of the host, Escherichia coli. We examined BrdUrd mutagenesis of rII mutants known to revert to wild type either by AT-to-GC or GC-to-AT transition pathways. While both reversion pathways were stimulated under all conditions analyzed, the AT-to-GC pathway was stimulated more when the E. coli reductase was functioning, while the GC-to-AT pathway was more specifically enhanced when the T4 reductase was active. These results confirm that ribonucleotide reductase is a determinant of BrdUrd mutagenesis, but our observations, plus experiments showing that BrdUrd has relatively small effects upon dNTP pool sizes, indicate that the relationship between deoxyribonucleotide metabolism and BrdUrd mutagenesis is more complex than anticipated.
Mol Gen Genet 1989 May
PMID:Ribonucleotide reductase: a determinant of 5-bromodeoxyuridine mutagenesis in phage T4. 267 47

Intracellular imbalances of dCTP produce both T----C transitions and an unusual class of transversions (A----C) at the aprt locus of CHO cells. Our data suggest that this transversion pathway is the consequence of dCTP:T mispairs which are not efficiently proofread during DNA replication.
Mol Cell Biol 1989 Apr
PMID:A novel pathway for transversion mutation induced by dCTP misincorporation in a mutator strain of CHO cells. 272 25

We previously demonstrated that 3'-azido-3'-deoxythymidine (AZT) inhibits growth proliferation of human bone marrow progenitor cells in vitro [Antimicrob. Agents Chemother. 31:452-454 (1987)]. The present study evaluates the effect of toxic concentrations of AZT on possible sites of toxicity in human bone marrow cells. Exposure of cells over a 6-hr period to AZT concentrations between 0.5 and 50 microM resulted in a decreased incorporation of tritiated deoxyguanosine into DNA. Unchanged AZT and its phosphorylated metabolites accumulated within cells after exposure to 10 microM [3H]AZT. 3'-Azido-3'-deoxythymidine-5'-monophosphate was the predominant metabolite, reaching a concentration of 49.2 +/- 14.1 pmol/10(6) cells after 48 hr, and a continuous increase was observed in all phosphorylated derivative levels between 2 and 48 hr of incubation. Using a highly sensitive and specific DNA polymerase assay, endogenous deoxyribonucleotide pool size(s) were analyzed for 48 hr after incubation of cells with a pharmacologically relevant concentration of 10 microM AZT. After a 6-hr exposure, 2'-deoxycytidine-5'-triphosphate and 2'-deoxythymidine-5'-triphosphate pools represented approximately 86 and 70% of the control values; levels returned to normal after 24 hr and remained subsequently unchanged. Nucleic acids of human bone marrow cells exposed for 24 hr to 10 microM [3H]AZT were purified and analyzed by cesium sulfate density gradient. No radioactivity was detected in the RNA region, whereas a significant amount was associated with the DNA region. Hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated specific incorporation of AZT into DNA. In additional studies, the amount of AZT incorporated into DNA was correlated with the initial extracellular AZT concentration. In particular, a significant relationship (p less than 0.0001) between the level of AZT incorporated into DNA and the inhibition of clonal growth was observed at concentrations of AZT between 1 and 25 microM (IC50 and IC85 for human bone marrow cells). In summary, these studies demonstrate that AZT is incorporated into DNA of human bone marrow cells and suggest that incorporation of AZT into DNA may be one mechanism responsible for AZT-induced bone marrow toxicity. In contrast, imbalance of deoxyribonucleotide pools by AZT appears unlikely to be associated with inhibition of DNA synthesis and toxicity in human bone marrow cells.
Mol Pharmacol 1989 Jul
PMID:Cellular pharmacology of 3'-azido-3'-deoxythymidine with evidence of incorporation into DNA of human bone marrow cells. 274 33

A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS-) is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [3H]uridine and [3H]thymidine. The CTPS- mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS- cells are deficient in CTP synthetase and thus are unable to convert [3H]UTP into [3H]CTP, which eventually is converted into [3H]dCTP and incorporated into DNA; (2) the growth of CTPS- mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [3H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 x 10(-7). The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [3H]uridine labeling assay. This modified "tritium suicide" method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement.
Somat Cell Mol Genet 1989 Jan
PMID:Single-step selection of mammalian cell mutants deficient in CTP synthetase. 291 65

The effects of inhibitors of poly(ADP-ribose) synthesis on cell growth and several parameters of nucleotide metabolism have been determined. At concentrations which produced similar inhibitions of poly(ADP-ribose) synthesis, 3-acetylaminobenzamide (1 mM) had no effect on L1210 cell growth, 3-aminobenzamide (5mM) was slightly inhibitory and 3-methoxybenzamide (5 mM) was a potent inhibitor of growth. During a 2-h incubation, none of the inhibitors affected ribo- or deoxyribonucleotide concentrations in cells treated with or without N-methyl-N-nitrosourea; however, N-methyl-N-nitrosourea treatment reduced dCTP concentrations by 50%. During a 24-hr incubation, 3-aminobenzamide and 3-acetylaminobenzamide did not lower ribonucleotide concentrations in cells grown with either undialyzed or dialyzed serum. In contrast, 3-methoxybenzamide caused a depletion of UTP in cells grown with undialyzed serum and caused a depletion of all purine and pyrimidine ribonucleotides in cells grown with dialyzed serum. 3-Aminobenzamide and 3-acetylaminobenzamide had no effect on the conversion of hypoxanthine to ATP and GTP but did slightly inhibit incorporation of formate into ATP and GTP. 3-Methoxybenzamide inhibited incorporation of both hypoxanthine and formate into purine ribonucleotides. 3-Aminobenzamide, 3-acetylaminobenzamide, and 3-methoxybenzamide all inhibited glycine incorporation into ATP and GTP and reduced both the incorporation of thymidine into DNA and the apparent specific activity of the dTTP pool. We conclude that inhibition of poly(ADP-ribose) synthesis causes little or no growth inhibition and has no effect on purine or pyrimidine nucleotide synthesis de novo. The effect of all the inhibitors on glycine and formate metabolism may be related to an inhibition of ADP-ribose synthesis or may be a secondary effect of the inhibitors. The growth inhibition and the reduction in nucleotide concentration caused by 3-methoxybenzamide are apparently secondary effects of this drug and may result from an inhibition of phosphoribosyl pyrophosphate synthesis.
Mol Pharmacol 1985 Aug
PMID:Specificity of inhibitors of poly(ADP-ribose) synthesis. Effects on nucleotide metabolism in cultured cells. 299 39

A physical map of safflower (Carthamus tinctorius L.) chloroplast DNA has been generated using SalI, PstI, KpnI and HindIII restriction endonucleases. Southern blots to single and double digests by these enzymes were hybridized with 32P-dCTP nick-translated KpnI probes, which were individually isolated from agarose gels. The plastid genome was found to be circular (151 kbp), to contain a repeated sequence of about 25 kbp, and to have small and large single copy regions of approximately 20 and 81 kbp, respectively. Heterologous probes from spinach and Euglena containing psbA, rbcL, atpA or rrnA structural genes were also hybridized with such single and double restriction enzyme digests and mapped on this circular chloroplast genome. The genetic map was found to be co-linear with that of spinach and many other higher plants.
Mol Cell Biochem 1985 Oct
PMID:The chloroplast genome of Carthamus tinctorius. 300 1

The deoxyuridine triphosphate nucleotidohydrolases (dUTPases, EC 3.6.1.23) from Escherichia coli K-12-,Acholeplasma laidlawii B-PG9-, human KB cell-, and the herpes simplex virus (HSV) type 1- and 2-induced dUTPases were purified and used to determine the effect of various mercury (II) compounds on their activities. Mercuric acetate, 5-mercuri-dUTP (HgdUTP), and 5-mercuri-dCTP (HgdCTP) acted as irreversible active site-directed inhibitors of the dUTPases purified from eukaryotic organisms but not those from prokaryotic organisms. The inhibition constants (Ki) were estimated for the KB, HSV-1, and HSV-2 dUTPases to be 8 +/- 2, 12 +/- 3, and 9 +/- 2 microM for mercuric acetate, 204 +/- 25, 121 +/- 15, and 111 +/- 10 microM for HgdUTP, and 775 +/- 25 and 651 +/- 23 microM for HgdCTP, respectively. The conversion of HgdUTP to its mercurithio-derivative resulted in a decrease in the affinity of the derivative for the eukaryotic dUTPases. The 5-mercurithioethylene glycol derivative of dUTP did not act as a substrate for the KB dUTPase but it did act as a substrate for the HSV-1- and HSV-2-induced dUTPases with Ki values of 526 +/- 47 and 483 +/- 32 microM, respectively. These results demonstrate that the eukaryotic dUTPases can be distinguished based upon differences in their affinities for the mercurithio-derivatives of dUTP and suggest that there are differences in the steric binding properties of the nucleotide-binding site of these enzymes.
Mol Pharmacol 1986 Mar
PMID:Effects of mercury (II) compounds on the activity of dUTPases from various sources. 300 36

We have isolated a highly enriched preparation of the multienzyme complex which synthesizes deoxyribonucleoside triphosphates (dNTPs) from bacteriophage T4-infected bacteria. By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparations. The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exist in vivo. The purified complex catalyzes the efficient five-step conversion of dCTP to dTTP. Experiments with several T4 mutants have demonstrated that gene products encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required. We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex. However, the dNTP-synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.
J Mol Recognit 1988 Feb
PMID:T4 phage deoxyribonucleoside triphosphate synthetase: purification of an enzyme complex and identification of gene products required for integrity. 307 39

From among a series of stable, aphidicolin-resistant mutant strains of mouse teratocarcinoma, derived from a multipotent parental line (PSA-1-80), three were selected for further study on the basis of their comparatively high degrees of resistance and elevated frequencies of spontaneous forward mutation to 6-thioguanine and ouabain resistance. Fluctuation tests confirmed that they were mutator strains. Since each of the three mutants was isolated after multiple rounds of selection, and since a variety of biochemical abnormalities were observed, it is likely that a number of mechanisms, probably consisting of overlapping subsets, determine the phenotypes. Abnormalities in the metabolism of the nucleotide substrates for polymerization are likely to be of major importance in mutants designated Aph-2 and Aph-3, as there were marked alterations in the dCTP and dATP pool sizes. The specific activity of DNA polymerase alpha was also increased. For the case of Aph-3, which exhibited the greatest (400-fold) increase in resistance to aphidicolin, a mutation in the structural gene for DNA polymerase alpha may be an additional important component, since in vitro assays revealed that the isolated enzyme was resistant to aphidicolin. For the case of Aph-1 however, only minor alterations in dNTP pools were observed, and there was no increase in the specific activity of DNA polymerase alpha or in the aphidicolin resistance of the isolated DNA polymerase alpha, suggesting yet another mechanism(s) underlying the aphidicolin resistance/mutator phenotype. All three mutants formed subcutaneous tumors in syngeneic mice; both Aph-1 and Aph-2 were multipotent; whereas Aph-3 was nullipotent.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Gen Genet 1987 Jun
PMID:Aphidicolin-resistant mutator strains of mouse teratocarcinoma. 311 23


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