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 intracellular deoxyribonucleoside triphosphate pools in mammalian cells affect diverse biological functions including the spontaneous or induced mutability. We have isolated from murine T-lymphosarcoma S49 cells, a mutant that is unable to convert dCMP to dUMP, contains deranged intracellular dNTP pools, and exhibits a mutator phenotype. The enzymatic defect in araC-6-1 cells is a deficiency of deoxycytidylate deaminase, which accounts for the high dCTP and low TTP intracellular pools. The addition of increasing concentrations of exogenous thymidine to araC-6-1 cells alters these dNTP pools in a predictable manner: increasing the TTP and diminishing the dCTP. Concomitant with this reversal of the dCTP:TTP ratio is a marked decrease in the mutation rate followed by an increase in the mutation rates at higher exogenous thymidine concentrations. This response of the mutation rate is in contrast to that seen in the control cell line containing normal deoxycytidylate deaminase. In the latter case, increasing thymidine concentration induces an enhanced mutation rate that parallels the later phase of the thymidine-induced mutation rate in araC-6-1 cells. The deficiency of deoxycytidylate deaminase, the endogeneous dNTP pool alterations, and the mutator phenotype of araC-6-1 cells are all recessive traits in cell-cell hybrids. These observations allow one to predict whether exogenous thymidine will be mutagenic, antimutagenic, or both for a given cell line and provide a basis for understanding conflicting reports in the literature concerning the effects of the thymidine on genomic stability.
Somat Cell Mol Genet 1985 Sep
PMID:The effects of exogenous thymidine on endogenous deoxynucleotides and mutagenesis in mammalian cells. 387 1

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4-6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653-664). Following a 30 s incubation with [3H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5' ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared with 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP, rUTP (0.1mM), as well as high levels of rATP (5mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.
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PMID:The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells. 617 44

Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating substrate concentration to initial velocity was changed from a sigmoidal shape to a hyperbolic one by the activator dCTP, while the inhibitor TTP increased the sigmoidicity of the curve. The molecular weight of deoxycytidylate deaminase was unchanged in the presence of allosteric effectors, indicating that aggregation-disaggregation is not the basis of regulation. Deoxycytidylate deaminase exhibited the greatest affinity for the substrate dCMP, with lesser affinity for ara-CMP, and least affinity for CMP. Ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 4 microM. These data indicate that human neoplastic cell deoxycytidylate deaminase is a highly regulated allosteric enzyme, which is likely to have a significant influence on cellular dUMP, dCTP and TTP pools. These findings further suggest, that the enzyme through its influence on dUMP levels is likely to modulate the biochemical effects of pyrimidine antimetabolites active against the thymidylate synthetase reaction and in the presence of elevated dCTP pools will promote deamination of ara-CMP to the inactive ara-UMP.
Mol Cell Biochem 1983
PMID:Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells. 658 81

We investigated the mechanism of action of 2-aminopurine (Apur) in eucaryotic cells. By analogy with studies in procaryotic systems, the base analog is presumed to incorporate into DNA predominantly opposite T where, upon subsequent DNA replication, it can mispair with C, inducing an A:T leads to G:C transition. This model predicts that Apur-induced mutagenesis will be enhanced by factors that favor formation of Apur-C mispairs, e.g., high levels of dCTP or low levels of TTP. We describe the use of a mutant T-lymphosarcoma cell line, AraC-6-1, which has an abnormally high dCTP pool and a low TTP pool, to test this prediction. AraC-6-1 cells were three- to fivefold more mutable by Apur than their parental cell line, NSU-1. This enhanced mutability by Apur could not be explained by altered incorporation of 3H-labeled Apur, by generally impaired ability to repair DNA damage, or by a direct effect of Apur on the endogenous deoxynucleotide pools. The addition of 10 microM thymidine to the growth medium of AraC-6-1 cells lowered their high dCTP pool (two- to threefold), raised the TTP pool (two- to threefold), and abolished their enhanced mutability by Apur. Further manipulation to produce an abnormally high TTP/dCTP ratio suppressed Apur-induced mutagenesis (8- to 10-fold) in both AraC-6-1 and NSU-1 cells. These observations support the hypothesis that Apur induces A:T leads to G:C transitions in mammalian cells by a mispairing mechanism.
Mol Cell Biol 1982 Sep
PMID:Mechanism of 2-aminopurine mutagenesis in mouse T-lymphosarcoma cells. 698 47

Human thymidine kinase TK1 isoenzyme has been purified 1800-fold from placenta to a specific activity of 2.9 nmoles/min/mg of protein. The rapid purification procedure includes affinity chromatography on a thymidine-Sepharose column. At all stages of purification, the enzyme showed irreversible lability. The native molecular weight was determined to be 45000. Human placental TK1 exhibited specificity for ATP and thymidine as substrates, and significant inhibition was found only with thymidine nucleotides. TTP was the most effective inhibitor.
Mol Cell Biochem 1982 Jun 11
PMID:Human thymidine kinase: purification and some properties of the TK1 isoenzyme from placenta. 698 69

To investigate whether DNA replication in malignant cells deviates from that of normal cells we compared DNA polymerases alpha, delta, and epsilon from normal rat liver to the enzymes from fast-growing (malignant) Novikoff hepatoma cells. DNA polymerases were purified 300-fold by three chromatographic steps. Characterization included measurement of physicochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), quantitation of catalytic activities using specific DNA primer templates (Km values) and inhibitors (Ki values), and identification of polypeptides which are strongly associated with DNA polymerases. Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. In contrast, the DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with the specific primer templates gapped calf thymus DNA and poly(dA.dT), were higher. Furthermore, sedimentation and diffusion coefficients, Stokes' radii, and frictional coefficient ratios of DNA polymerases alpha and epsilon from malignant cells significantly deviated. In addition, when the dNTP-binding sites were probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP), significantly lower Ki values were obtained for the polymerases from Novikoff cells indicating lower affinity of the dNTP binding site to deoxyribonucleoside 5'-triphosphates. Altered catalytic and molecular properties are possibly a consequence of malignant transformation. It is to be expected that similar changes occur in DNA polymerases of other tumors. In particular, diminished affinity to primer templates and weakened nucleotide binding leads to lowered specificity of nucleotide selection in the base-pairing process and is therefore likely to cause an enhanced mutation rate during malignant progression.
J Mol Med (Berl) 1995 May
PMID:DNA polymerases alpha, delta, and epsilon of Novikoff hepatoma cells differ from those of normal rat liver in physicochemical and catalytic properties. 767 Sep 30

We have generated transgenic mice that express the simian virus 40 (SV40) large T antigen under the control of a 1109 bp 5'-flanking sequence of the human thyrotropin beta-subunit (TSH beta) gene. The hybrid gene, termed TTP-1, was microinjected into fertilized mouse eggs and 11 transgenic mice were obtained. One of the transgenic mice, a female, a phenotypical dwarf, developed a pituitary tumor and wasted away from 7 to 9 weeks after birth. To establish the transgenic mouse line, her ovaries were transferred to a normal female, whose ovaries were removed beforehand. To examine the tissue specificity of transgene expression, mRNA of SV40 large T antigen was monitored in various tissues from the transgenic mice by the reverse transcriptase-polymerase chain reaction analysis, and was detected only in the pituitary. Histological and immunohistochemical analyses showed that the pituitary tumors of the transgenic mice were composed of poorly differentiated pituitary cells expressing SV40 large T antigen. These results indicated that the 1109 bp sequence of the human TSH beta 5'-flanking region is essential for pituitary-specific expression of SV40 large T antigen in transgenic mice, which exhibited a dwarf phenotype and developed pituitary tumors. The tumors were composed of undifferentiated cells and did not produce thyrotropin. These transgenic mice should provide a valuable animal model for studying the pathogenesis of anterior pituitary tumors.
Mol Cell Endocrinol 1994 Nov
PMID:Targeted pituitary tumorigenesis using the human thyrotropin beta-subunit chain promoter in transgenic mice. 785 21

Two acidic chitinase isoforms, SP1 and SP2, have been purified to homogeneity from leaves of sugar beet (Beta vulgaris) infected with Cercospora beticola. SP1 and SP2 are extracellular proteins with an apparent molecular mass of 35 kDa and an approximate pI of 4.2. Since the only major difference was slightly diverging M(r)'s, only the SP2 chitinase was further characterized. Partial amino acid sequence data for SP2 was used to generate a polymerase chain reaction (PCR) clone employed for the isolation of a cDNA clone encoding SP2. SP2 exhibits significant structural identity with the class IV chitinases from sugar beet, rapeseed, bean and maize, but differs from the other members of this class in having a longer hinge region, comprising 22 amino acid residues, with a repeated 'TTP' motif. Western blotting analyses, using antibody raised against SP2, demonstrated an induction of SP protein during infection with C. beticola. The induction was very local, with high protein accumulation found close to the infection site only. Amino acid compositional analysis of SP2 revealed that five out of fourteen prolines are hydroxylated. No glucosamine or galactosamine residues are present. Evidence was obtained that SP2 is glycosylated with a limited number (< or = 7) of xylose residues: (1) SP2 was stained with the periodic acid-Schiff (PAS) reagent, (2) electrospray mass spectrometry on SP2 gave a series of M(r)'s with a consistent increase between two molecular masses of 132 Da, (3) SP2 was recognized by an antibody specific for beta-1,4-D-xylopyranose. The vacuolar class I chitinases A and B in tobacco have recently been shown to comprise a new class of hydroxyproline-containing proteins (Sticher et al., Science 257 (1992) 655-657). The SP2 chitinase differs from these in being glycosylated and, thus, represents a novel type of hydroxyproline-containing glycoproteins in plants.
Plant Mol Biol 1994 May
PMID:A hydroxyproline-containing class IV chitinase of sugar beet is glycosylated with xylose. 801 73

Wheat germ DNA polymerase A, a gamma-like enzyme, recognized efficiently natural and synthetic RNA templates, resembling a retroviral reverse transcriptase (P. Laquel et al., Biochim Biophys Acta 1048 (1990): 139-148). Ammonium-21-tungsto-9-antimoniate (HPA-23), an antiviral drug, inhibited the DNA polymerase A activities, independently of the template primers used, i.e. activated DNA or polyriboadenylic acid oligodeoxythymidylate (poly(rA)-oligo(dT)). The inhibition observed in the poly(rA)-oligo(dT)-directed DNA polymerase A activity occurred in the presence of either Mg2+ or Mn2+ as divalent cation, and also with the 2'-fluoro analogue of poly(rA) as template. HPA-23 was a non-competitive inhibitor with respect to TTP, activated DNA, poly(rA)-oligo(dT), and poly(dAfl)-oligo(dT). A preincubation study showed a reversible HPA-23 binding to DNA polymerase A, in the presence of poly(rA)-oligo(dT) as the template primer.
Plant Mol Biol 1993 Dec
PMID:Inhibition of the wheat germ DNA polymerase A activity by the antiviral drug HPA-23. 826 Jun 25

We have previously reported the purification of a 37 KDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura and have shown that it is present in a subset of TTP patients, but absent in normal subjects. In this study, we would like to report some of the physico-chemical and immunological properties of this protein. The native molecular weight of PAP p37 from gel filtration was found to be 36,000, which is in agreement with denatured molecular weight (37,000), determined by SDS--polyacrylamide gel electrophoresis under both reducing and non-reducing conditions. The values of Stoke's radius (25A), diffusion coefficient (8.59 x 10(-7)cm2/s) and frictional ratio (1.13), determined by molecular sieve chromatography, suggest that the native protein is compact and globular. The purified protein has an S20,w of 3.5s. Preliminary carbohydrate analysis suggested that p37 is a glycoprotein and contained about 11% neutral sugars and 6.6% sialic acid. Amino acid analysis indicated that the protein is relatively rich in aspartate and serine and has low cysteine, methionine and tryptophan contents. In dot immunobinding ELISA assay, PAP p37 did not react with antibodies to thrombospondin, fibrinogen, fibronectin, plasminogen and von Willebrand factor. Our results suggest that PAP p37 is a single polypeptide compact and globular glycoprotein and is immunologically not related to the aforementioned proteins.
Biochem Mol Biol Int 1993 Jun
PMID:Characterization of platelet agglutinating protein p37 purified from the plasma of a patient with thrombotic thrombocytopenic purpura. 836 16


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