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)

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

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.
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PMID:Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate. 666 44

Antibodies to homogeneous calf thymus DNA polymerase-beta and calf thymus DNA polymerase-alpha preparations were raised in rabbits. The antiserum against calf thymus DNA polymerase-beta cross-reacts with all vertebrate DNA polymerase-beta preparations tested, but does not cross-react with trypanosome DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and Escherichia coli DNA polymerase I. The antibodies against calf thymus DNA polymerase-alpha cross-react with DNA polymerase-alpha from mouse, human, and chicken, but do not cross-react with DNA polymerase-alpha from sea urchin embryos and Drosophila embryos, DNA polymerase-beta, DNA polymerase-gamma, terminal transferase, yeast DNA polymerases, and E. coli DNA polymerase I.
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PMID:Immunological reagents for comparisons of DNA polymerase-alpha and DNA polymerase-beta. 677 65

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

The extensive breakdown of immune homeostasis in the motheaten mouse (me/me) has been ascribed to a single gene defect on chromosome 6 (ref. 1). These mice develop skin lesions within the first week of life, do not thrive, and die within the first 3--8 weeks. There is severe hypergammaglobulinaemia with multiple species of circulating autoantibody and deposition of immune complexes in the thymus, skin, lungs and kidneys. A single gene defect producing such catastrophic results may provide an important model for understanding autoimmune phenomena. We report here a virtual absence of terminal deoxynucleotidyl transferase-positive (TdT+) cells in the bone marrow, thymus and spleen of motheaten mice. TdT is a DNA polymerase which has the unique capacity to polymerize nucleotides in the absence of template direction. Although no in vivo biological function of this enzyme has been established, its unique appearance in the bone marrow and thymus of adult mammals and its in vitro biochemical activity have led to a proposed role for TdT in the somatic diversification of lymphocytes. Bone marrow TdT+ cells have been shown to belong to both T and B cell populations and may also include precursor cells common to these lineages. Although the role of TdT in the acquisition of appropriate T- and B-cell specificities is not known, our results are the first to correlate the virtual absence of TdT+ cells with a severe autoimmune syndrome. We investigated the level of TdT+ cells in neonatal me/me mice and their normal littermates and the susceptibility of TdT+ cells to circulating autoantibody in motheaten mouse serum.
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PMID:Deficiency in cells expressing terminal transferase in autoimmune (motheaten) mice. 721 27

The decrease of functional capacity of cellular immunity during ageing seems to be due to cellular changes of stem cells, particularly in the growth properties and the cell density in T-cell subsets. We approached this problem at the molecular biological level by quantifying the key enzymes necessary for DNA synthesis in bone marrow cells from mice: deoxynucleotidyl transferase (TdT) and DNA polymerase alpha. The bone marrow cells were fractionated on a discontinuous bovine serum albumin density gradient and the extractable enzyme activities (expressed per 10(8) nucleated cells in the respective fraction) were determined. TdT activity was found to decrease markedly during ageing. Mature animals contain only 34% and senescent animals only 13% of the activity observed in immature mice. From the density distribution analysis it was found that a shift of TdT-containing cells to the lower density occurs. The specific DNA polymerase alpha activity also decreases in bone marrow cells with age. While the overall activity amounts in immature cells to 78 enzyme units/10(8) cells, it decreases in mature cells to 57 units/10(8) cells, and in cells from senescent animals to 36 units/10(8) cells. Density distribution analysis of the cells shows that the highest activity is observed in the low-density fraction. From these experimental data we conclude that in the fractions containing precursor T-cells, a reduced number of proliferating cells is present.
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PMID:Age-dependent alterations of DNA synthesis. Terminal deoxynucleotidyl transferase and DNA polymerase activities in bone marrow subpopulations from mice. 743 1

Macronuclear telomeres in Oxytricha exist as DNA-protein complexes in which the termini of the G-rich strands are bound by a 97-kDa telomere protein. During telomeric DNA replication, the replication machinery must have access to the G-rich strand. However, given the stability of telomere protein binding, it has been unclear how this is accomplished. In this study we investigated the ability of several different DNA polymerases to access telomeric DNA in Oxytricha telomere protein-DNA complexes. Although DNA bound by the telomere protein is not degraded by micrococcal nuclease or labeled by terminal deoxynucleotidyltransferase, this DNA serves as an efficient primer for the addition of telomeric repeats by telomerase, a specialized RNA-dependent DNA polymerase (ribonucleoprotein reverse transcriptase), EC 2.7.7.49. Moreover, in the presence of a suitable complementary C-rich DNA template, AMV reverse transcriptase and the E. coli Klenow fragment will also elongate DNA bound by the telomere protein. These findings indicate that the 3' terminus and the Watson-Crick base pairing positions are exposed in the protein complex. We propose that the telomere protein can serve a dual role at the telomere by protecting the DNA phosphate backbone from degradation while simultaneously exposing the DNA bases for replication.
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PMID:DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases. 750 21

We present a second-strand cDNA synthesis method that takes advantage of both the very high processivity and the very high 3' exonuclease activity of T7 DNA polymerase. The first strand is synthesized with reverse transcriptase using oligo(dT) as a primer. After alkaline hydrolysis of the mRNA template, a tract of dT residues is synthesized with terminal transferase at the 3' end of the first strand. The second strand is synthesized using oligo(dA) as a primer. Several oligo(dA) molecules probably anneal to the poly(dT) tract. Because the 3' exonuclease activity of T7 DNA polymerase is very high, the region of the tract annealed to these oligo(dA) molecules is digested. However, the region of the tract annealed to the very oligo(dA) molecule used as a primer for second-strand synthesis is protected. The resulting cDNA molecules could be cloned with a high efficiency. The size distribution of cloned c-myc DNAs was estimated by Southern blot analysis of phage DNA prepared from the amplified library and by analysis of isolated clones. The results indicate that this method allows to obtain full-length cDNA clones with a high efficiency.
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PMID:Efficient second-strand cDNA synthesis using T7 DNA polymerase. 752 64

Programmed cell death (PCD) via apoptosis is characterized by nuclear pyknosis and fragmentation, and biochemically by oligonucleosomal cleavage of DNA. Apoptosis occurs in the developing nervous system, whereas its role in neurodegenerative diseases is still debated. Recognition of apoptotic cells has recently been facilitated by in situ end-labeling (ISEL) techniques which identify DNA strand breaks through incorporation of labeled nucleotides. We have applied two ISEL assays to physiological and pathological conditions affecting the nervous system in which PCD is likely to occur. Terminal transferase assay was more sensitive than DNA polymerase assay and allowed the recognition of a larger number of cells than conventional histology. Apoptotic cells were readily found in the developing spinal cord and dorsal root ganglia. Medulloblastomas, gliomas, brain lymphomas and metastases showed abundant apoptotic cells either isolated or grouped in small foci. Labeling was also found in cells without a clearcut apoptotic morphology. Apoptotic cells were not found in Alzheimer's disease, amyotrophic lateral sclerosis and human and mouse prionic encephalopathies. Our results show that ISEL is a useful technique for demonstrating apoptotic cells in nervous tissue during development and in brain tumors. Lack of staining in neurodegenerative diseases suggests that other types of PCD might be involved.
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PMID:A study of apoptosis in normal and pathologic nervous tissue after in situ end-labeling of DNA strand breaks. 752 80

The structure of new nucleoside--3'-nitro-2',3'-dideoxythymidine (NIT) possessing moderate anti HIV activity in MT-4 cell culture was investigated by X-ray analysis. These data showed that conformation of NIT in crystal is similar to that of one of crystallographically independent forms of 3'-azido-2',3'-dideoxythymidine. 3'-Nitro-2',3'-dideoxythymidine 5'-triphosphate (NITTP) was synthesized and its ability to inhibit human and viral DNA polymerases was studied. NITTP proved to be effective and highly selective terminating substrate of DNA synthesis catalyzed by HIV and AMV reverse transcriptases. Human DNA polymerase alpha as well as DNA polymerase beta (rat liver), terminal deoxynucleotidyltransferase (calf thymus) or HSV-1 and CMV DNA polymerases did not incorporate NITTP into a growing DNA chain.
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PMID:[Conformation of crystalline 3'-nitro-2',3'-dideoxythymidine and properties of its 5'-triphosphate as a terminator substrate of retroviral reverse transcriptase]. 754 Feb 54


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