Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have addressed the possibility of terminal transferase involvement in somatic mutagenesis and the creation of N-region diversity, by measuring the ability of TdT to enhance single-base substitution mutagenesis during in vitro DNA synthesis. Using 3 independent assays we find that terminal transferase produces only a small increase in base-substitution mutagenesis when assayed in the presence of DNA polymerase-beta. In the presence of either polymerase-alpha or E. coli polymerase-I, however, no detectable increase in TdT-induced mutagenesis is seen. Furthermore, in an assay capable of detecting a variety of mutational events, terminal transferase primarily produces complex addition/deletion mutations, as well as a few multiple, tightly-clustered, single-base mutations. We conclude that the majority of the scattered single-base changes that occur during antibody gene differentiation are not catalyzed by terminal transferase, but instead result from another error-prone DNA synthetic process (possibly utilizing DNA polymerase-beta).
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PMID:Base substitution mutagenesis by terminal transferase: its role in somatic mutagenesis. 295 4

Until recently, lineage fidelity was thought to be preserved in leukaemic cells, which by available tests showed surface markers and enzymatic patterns characteristic of an appropriate normal cell lineage and stage of differentiation. Our data indicate that this theory is too restrictive. If leukaemogenesis occurs in pluripotent progenitors in a relatively high percentage of cases, we would propose a model in which lymphoid and myeloid differentiation antigens are expressed simultaneously until the progenitor cell commits to a single lineage. Lineage commitment could involve external factors, e.g. growth factors (Sherr et al, 1985), that cause genes specific for the opposite lineage to be 'switched off'. The control of gene expression in mammalian cells and the specific chromosomal sites of genes coding for the various lineage-associated markers remain uncertain. However, recent studies indicate that most, if not all, leukaemic cells contain chromosomal abnormalities, many involving rearrangements of DNA (Williams et al, 1986). Since the control of eukaryotic gene expression is known to involve numerous sequence elements, some acting at a distance from the site of transcription (Dynan and Tjian, 1985), genetic perturbations within the cell (e.g. a reciprocal translocation) could be expected to deregulate certain genes, leading to their under- or overexpression analogous to activation of the c-myc oncogene by the 8;14 translocation in Burkitt's lymphoma. Thus, an almost infinite variety of cell lineage-related phenotypes could be expected from this mechanism alone, even if the transforming event did not involve a pluripotent stem cell. Also, we have hypothesized that enzymes such as TdT, a DNA polymerase that catalyses polymerization of deoxyribonucleotides without a DNA template, could serve as a modifier of DNA sequences, permitting otherwise inactive genes to be expressed (Stass and Mirro, 1985). It is interesting that most cases of childhood acute mixed-lineage leukaemia are TdT positive, even though this is not true for the chronic leukaemias of adults. It is now clear that unusual combinations of myeloid and lymphoid cell lineages are much more common in acute leukaemia than have been generally recognized or suspected. The traditional division of the acute leukaemias into ALL and AML may not be the most accurate way to represent this class of haematological malignancies. That mixed-lineage leukaemia may require alternative therapy is a clinically important observation and underscores the need for comprehensive testing of blast cells at diagnosis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Lineage heterogeneity in acute leukaemia: acute mixed-lineage leukaemia and lineage switch. 353 42

We evaluated a newly developed solid-phase immunoassay (EIA) of terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.3) and compared it with the enzymatic assay of TdT involving DNA polymerase. We assessed the precision, performance characteristics, and clinical efficacy of the EIA procedure, using 249 specimens of peripheral blood and bone marrow and 118 specimens of whole blood. On linear regression analysis of results for these 249 samples as measured by the two procedures, the correlation coefficient was 0.87. Distribution of TdT in mononuclear cells isolated from whole blood and bone marrow of subjects in several disease categories indicated good concordance between the two assay procedures. The EIA procedure is precise, can be performed on whole blood without first isolating mononuclear cells, is nonisotopic, and shows potential as a quantitative indicator for the differential diagnosis and monitoring of human leukemia.
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PMID:Solid-phase enzyme immunoassay of terminal deoxynucleotidyl transferase evaluated. 354

Terminal deoxynucleotidyl transferase (EC 2.7.7.31) is a eucaryotic DNA polymerase that does not require a template. The tryptophan environments in calf thymus terminal transferase were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was separated by time-resolved emission spectroscopy. Nanosecond fluorescence decays at 296-nm excitation and various emission wavelengths were deconvolved by global analysis, assuming that the lifetimes but not the relative weighting factors were independent of emission wavelength. The data were fit to three exponentials of lifetimes tau 1 = 1.4 ns, tau 2 = 4.5 ns, and tau 3 = 7.7 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 328, 335, and 345 nm. The accessibility of individual tryptophan environments to polar and nonpolar fluorescence quenchers was examined in steady-state and time-resolved experiments. In the presence of iodide and acrylamide, the steady-state emission spectra shift to the blue. However, at low quencher concentrations, the emission from the 7.7-ns component (maximum 345 nm) is hardly affected, suggesting that this hydrophilic tryptophan environment is buried within the protein. On the other hand, the red shift in the steady-state emission spectrum in the presence of trichloroethanol indicates that the 1.4-ns component (maximum 328 nm) is an exposed hydrophobic tryptophan environment. The results are consistent with an inside-out model for terminal transferase protein, with the more hydrophobic tryptophan(s) near the surface and the most hydrophilic tryptophan(s) in the core.
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PMID:Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra. 408 79

Fourteen streptovaricin derivatives were tested for inhibition of cellular nucleotide polymerases (deoxyribonucleic acid polymerases alpha, beta, and gamma, terminal deoxynucleotidyltransferase [TdT], and ribonucleic acid polymerase II), simian sarcoma virus deoxyribonucleic acid polymerase, and herpes simplex virus type 1-induced deoxyribonucleic acid polymerase (HSV-DP). Three compounds (strep-tovadienal C, prestreptovarone, and streptoval Fc) preferentially inhibited TdT and HSV-DP over the other enzymes. These compounds inhibited HSV-DP more potently than they inhibited TdT. Evidence indicated that the mode of inhibition of TdT and HSV-DP by streptovadienal C and prestreptovarone was by interaction with the enzymes and not with template-primer, initiator, substrates, or divalent cations required for enzyme activity. Furthermore, data suggested that these compounds bind with greater affinity to HSV-DP than to TdT. Streptovadienal C and prestreptovarone were examined for their effect on the replication of herpes simplex virus type 1 in African green monkey kidney (CV1) cells. These compounds produced 2- and 3-log drops in virus titer, respectively, at concentrations not significantly affecting cell viability. This correlated with evidence indicating a greater binding affinity of these compounds for HSV-DP over cellular nucleotide polymerases.
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PMID:Inhibition of cellular and virus-associated nucleotide polymerases by, and anti-herpes simplex virus activity of, streptovaricin derivatives. 611 56

The ubiquitous trace metal zinc has been discovered since a long time as an intrinsic element in all biological systems. However, its role other than structural or catalytic in enzymes is poorly defined. Zinc plays a determinative role both in primary and secondary T lymphocyte production. Experimental data support the notion that during intrathymic maturation, non-autoreactive, immunocompetent T cell clones are selected from the excess of immature thymocytes as a result of expansion of bone marrow derived prothymocytes in response to pleiotropically acting alarmon (s) and a subsequent escape via the thymic stroma cells from nucleotide-mediated "biochemical suicide". The activity of alarmon (Ap4A), nucleotide metabolizing enzymes (TdT, DNA polymerase, thymidine kinase, 5'-nucleotidase) and some of the soluble stromal cell products (FTS) require constitutive zinc. In the peripheral lymphoid organs the magnitude and duration of antigen induced, T cell mediated immunoreactions are regulated by T-cell growth factor (IL-2). Using receptor specific monoclonal antibody probes, it has been established recently that the intracellular role of IL-2 is probably to induce the phenotypic expression of high affinity transferrin receptors, known to be the main zinc transporter system in T-lymphocytes. The coordinative role of zinc in T lymphocyte development via the inducible metallothionein system is emphasized. Some clinical aspects of zinc metabolism are discussed.
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PMID:Zinc and immunity. 623 34

The biochemical activity of terminal transferase (TdT) in the thymocytes of leukemic AKR mice has no relationship to cell cycle stage, unlike the activity of replicative DNA polymerase which increases during the period of DNA synthesis. Moreover, such assays of DNA polymerase alpha reveal a shift in enzyme activity from cytoplasm to nucleus during S phase. In the present study, the role of TdT in DNA metabolism was explored further by examining the intracellular location of the enzyme during cytokinesis. Single cell suspensions of thymocytes from leukemic AKR mice were partially synchronized by velocity sedimentation in a sucrose gradient at unit gravity and harvested according to cell cycle stage. The content and location of TdT in individual cells was determined by indirect immunofluorescence using a rabbit antiserum to calf thymus TdT as the primary antibody. There was no relationship of fluorescence intensity or of the proportion of TdT-positive cells to cell cycle stage. In all samples examined (n = 6) the enzyme was located almost entirely in the nucleus throughout cytokinesis. These results do not support the hypothesis that the intracellular location of TdT may vary with cell cycle stage and a role for the enzyme in DNA synthesis remains to be defined.
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PMID:The intracellular location of terminal transferase does not vary with cell cycle stage. 661 60

We have synthetised 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate (in short : rATP-DNP), a derivative of ATP which carries a dinitrophenyl group. We show that rATP-DNP is a substrate for calf thymus deoxynucleotidyl terminal transferase (EC 2.7.7.31) and E. coli DNA polymerase I (Kornberg polymerase EC 2.7.7.7.). It can therefore be incorporated into DNA molecules by elongation from 3' ends or by nick translation. The incorporated dinitrophenyl group can be recognized by specific antibodies which can then be detected by anti-antibodies coupled to an enzyme. DNP groups could also be introduced into DNA after enzymatic incorporation of 8-aminohexyl adenosine 5' triphosphate and reaction with 1-fluoro-2-4-dinitrobenzene. Thus, DNA molecules carrying DNP groups can ultimately be revealed by enzymatic coloured reactions. Potential uses of this enzymatic labelling as a substitute to the radioactive detection of nucleic acids, are discussed.
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PMID:Synthesis of 8-(2-4 dinitrophenyl 2-6 aminohexyl) amino-adenosine 5' triphosphate: biological properties and potential uses. 675 67

Terminal deoxynucleotidyl transferase is a unique DNA polymerase that can carry out DNA synthesis on an initiator molecule in the absence of a template. The usefulness of this enzyme as a biological marker for following patients during treatment and remission has been suggested. The potential usefulness of this enzyme in predicting the onset of relapse before any morphological indications has been demonstrated in chronic myelogenous leukemia patients in blast phase of the disease. In order to be able to detect low levels of TdT activity especially during remission phase, we have used cell separation techniques which can enrich cell populations containing TdT activity. A number of cell separation techniques have been developed to separate different cell types. We have used the techniques of unit gravity sedimentation and free flow electrophoresis to achieve enrichment of TdT positive cell populations. Our results show that up to 20 fold enrichment of TdT activity in normal human bone marrow can be accomplished by using cell separation techniques. With the use of free flow electrophoresis, we have achieved enrichment of TdT positive cell populations from normal human bone marrow, cells from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia in blast phase of the disease. No TdT positive cells were detected in patients with acute myelogenous leukemia. These cell separation techniques should prove to be useful in early detection of relapse in patients in remission.
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PMID:Enrichment of terminal deoxynucleotidyl transferase activity by cell separation. 698 Dec 92

Calf thymus DNA polymerases alpha and beta [EC 2.7.7.7] and terminal deoxynucleotidyl transferase [EC 2.7.7.31] were analyzed on two-dimensional gel slabs. DNA polymerase beta appeared as a single spot on two-dimensional gel at the position of 40,000 daltons and pI 8.0 using non-equilibrium pH gradient gel electrophoresis for the first-dimensional run. By overlapping gel slabs, it was possible to identify the distinct spot of DNA polymerase beta among many polypeptide spots of a crude enzyme fraction. 10S DNA polymerase alpha showed two clusters of polypeptide spots on two-dimensional gel slab. One cluster was composed of three large polypeptides of 140,000-150,000 daltons and another was composed of four smaller polypeptides of 46,000-50,000 daltons. All these spots were arranged in a narrow pI range (6.5-6.8) although each spot showed a distinct pI value. Purified terminal deoxynucleotidyl transferase showed three polypeptides of 57,000, 42,000, and 33,000 daltons at similar pI values (7.0-7.2). Each polypeptide consisted of plural spots which differed slightly in pI but were the same in molecular weight. These results suggest a microheterogeneity of polypeptides of terminal deoxynucleotidyl transferase as well as those of 10S DNA polymerase alpha.
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PMID:Analysis of calf thymus DNA polymerases alpha and beta and terminal deoxynucleotidyl transferase on two-dimensional polyacrylamide gels. 713 Jan 50


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