Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using a restriction fragment length polymorphism which can distinguish the two copies of the thymidine kinase (tk) gene in the TK6 human lymphoblastoid cell line, we have identified heterozygous subclones with alternate active alleles. Quantitative mutagenesis studies with X-rays revealed a markedly different response, depending on which homolog carried the active allele. The slopes of the dose-response curves differed by approximately 10-fold for mutation of the two alleles and this relationship held true for several independently isolated cell lines. Only one of the cell lines showed a different response to ethyl methanesulfonate. There were no differences among any of the cell lines at the X-linked hprt locus. Analyses of TK- mutants recovered from these cell lines indicated that the reduced yield of mutants from the one allele may be due, at least in part, to a lack of a specific class of TK- mutant, that is, the slow-growing mutants which have been associated with large-scale mutagenic events.
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PMID:A comparison of induced mutation at homologous alleles of the tk locus in human cells. 167 26

TK6 human lymphoblast cells (tk +/-; hprt+) were treated with various concentrations of 2-amino-N6-hydroxyadenine (AHA) for 24 h. AHA was quite toxic to TK6 cells in the dose range 0-0.05 micrograms/ml, but additional toxicity was not observed between 0.05 and 0.10 micrograms/ml. AHA induced mutations at 2 distinct genetic loci: the autosomal thymidine kinase (tk) and the X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt). Significant levels of both tk-NG mutants (normal growth rate of 16-18 h, colonies visible after 10-11 days incubation) and tk-SG mutants (slow growth rate of greater than 24 h, colonies visible after 18 days incubation) were induced. 15 hprt- mutants were isolated and analyzed by Southern blot. 8 of these had normal restriction fragment patterns after digestion with PstI, EcoRI, and HindIII, and were defined as 'point' mutations; the remaining 7 had partial deletions of the hprt gene. 32 tk- mutants were also isolated. 3 of 22 normal growth mutants and 6 of 10 slow growth mutants had lost the active tk allele. These data suggest that both point mutations and larger-scale alterations are induced by AHA.
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PMID:Mutagenicity of 2-amino-N6-hydroxyadenine to TK6 human lymphoblast cells. 187 Jun 12

Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected. S-phase BHK-cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP-kinase. This complex was partially, but not completely, disrupted when lysates from GO-phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus-induced ribonucleotide reductase and a minor proportion of the HSV-thymidine kinase cosedimented rapidly. The virus-induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S-phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous "activated" DNA. Added DNA primer-template was required, however, for maximal activity of the polymerase in gradient fractions derived from GO-phase cells and from HSV-infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV-infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S-phase cells about 10 pmol/10(6) cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/10(6) cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV-infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.
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PMID:Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I. 244 4

Six murine L cell lines expressing five different human cell surface antigens have been prepared by DNA-mediated gene transfer. Ltk- cells were transfected with calcium phosphate co-precipitates of human genomic DNA and a plasmid containing the Herpes simplex thymidine kinase gene. After HAT selection, transfectants expressing specific cell surface antigens were identified by in situ immune rosetting using monoclonal antibodies. In this way, transfected cell lines expressing the CD9 antigen, the CD31 antigen (two lines), a unique platelet antigen, an X-linked antigen (R1), and a previously unreported monocyte antigen 11D1 were prepared. These cell lines have proved useful in the definitive assignment of monoclonal antibodies to specific CD groups. In addition, they provide a source of mRNA for use in expression cloning of the genes for these antigens.
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PMID:Transfection of genes for human cell surface antigens identified by monoclonal antibodies. 262 57

Chromosome-mediated gene transfer (CMGT) of the human genes for hypoxanthine phosphoribosyl transferase (HPRT) and cytosol thymidine kinase (TK1) into HPRT deficient mouse A9 cells or TK deficient Swiss mouse 3T3TK- cells was found to occur at frequencies at least one order of magnitude higher than DNA-mediated gene transfer (DMGT). The frequency of CMGT into 3T3TK- cells was reduced by more than an order of magnitude by a posttreatment of the recipient cells with dimethyl sulphoxide (DMSO). After CMGT, expression of the non-selected genes coding for galactokinase (GALK) and acid alpha-glucosidase (GAA), both syntenic with TK1, was observed in a number of transformants. From the pattern of cotransfer, a tentative gene ordering of CENTROMERE-GALK-TK1-GAA on human chromosome 17 was deduced. Chromosome-mediated cotransfer of X-linked human phosphoglycerate kinase (PGK) with HPRT was observed in two out of 33 A9 transformants analysed. DNA-mediated cotransfer of a syntenic gene was only observed for GALK, cotransferred with TK1 in two out of 18 TK+ transformants of mouse LTK- cells. Therefore, with murine cells as recipients of human donor genetic material, CMGT results in a higher frequency of transfer and a higher incidence of cotransfer of syntenic genes than DMGT using cellular DNA in the same cell system.
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PMID:Cotransfer of syntenic human genes into mouse cells using isolated metaphase chromosomes or cellular DNA. 388 35

The activities of TdR kinase2 (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21), AdR kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76), GdR kinase (ATP: deoxyguanosine 5'-phosphotransferase, without EC number), ATP (Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3), nucleoside diphosphatase (nucleoside diphosphate phosphohydrolase, EC 3.6.1.6), nucleoside phosphotransferase (AMP: deoxynucleoside phosphotransferase, EC 2.7.1.77) and ribonucleotide 5'-diphosphate reductase (EC 1.17.4.1) were assayed in mitochondria of normal and regenerating rat liver. The activities of deoxynucleoside kinases are regulated: (a) by feedback inhibition of TdR kinase with dTTP and dCTP, and GdR kinase with dGTP; (b) GdR and AdR kinases by AdR and GdR inhibition, respectively; (c) by stimulation of GdR kinase with dTDP, dTTP and dATP. The stimulatory effects are correlated with changes of ATP (Mg2+)-ase and NDP-ase activities in regenerating liver mitochondria.
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PMID:Regulation of deoxynucleoside kinase activities in rat liver mitochondria. 612 3

As a first step in the development of a multiple-marker, mammalian cell mutagenesis assay system, we have isolated a Chinese hamster ovary (CHO) cell line that is heterozygous for both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci. Presumptive aprt+/- heterozygotes with intermediate levels of APRT activity were selected from unmutagenized CHO cell populations on the basis of resistance to low concentrations of the adenine analog, 8-azaadenine. a functional aprt+/ heterozygote with approximately 50% wild-type APRT activity was subsequently used to derive sublines that were also heterozygous for the tk locus. Biochemical and genetic characterization of one such subline, CHO-AT3-2, indicated that it was indeed heterozygous at both the aprt and tk loci. CHO-AT3-2 cells permitted single-step selection of mutants resistant for 8-azaadenine or 5-fluorodeoxyuridine, allowing quantitation and direct comparison of mutation induction at the autosomal aprt or tk loci, as well as in the gene involved in ouabain resistance or at the X-linked, hypoxanthine--guanine phosphoribosyltransferase (hgprt) locus. Significant dose-dependent increases in mutation frequency were observed for all 4 genetic markers after treatment of CHO-AT3-2 cells with ethyl methanesulfonate.
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PMID:Mutagenicity testing in mammalian cells. I. Derivation of a Chinese hamster ovary cell line heterozygous for the adenine phosphoribosyltransferase and thymidine kinase loci. 644 63

Relatively little work has been done on the influence of the position of the cell in the cell cycle on ionizing radiation-induced mutagenesis. We synchronized WTK1 human lymphoblastoid cells with 200 microM lovastatin for 48 h; under these conditions more than 80% of the cells were arrested in G1 phase. Upon release, there was a 12-15-h lag followed by movement of a large fraction into S phase. We irradiated cells with either 1.5 Gy X rays at 1, 15, 18, 21 or 24 h or 1.5 Gy gamma rays at 1, 5, 10, 15 or 24 h after release from lovastatin. We showed that WTK1 cells were most sensitive to ionizing radiation-induced toxicity in G1 and into S phase, and more resistant in mid to late S and G2/M phase. Somewhat surprisingly, we found that the two different gene loci had different sensitivities to radiation-induced mutation through the cell cycle. Cells in late G1 through mid-S phase were most sensitive to radiation-induced mutations at the autosomal thymidine kinase (TK) locus, whereas G1 phase was the most sensitive phase at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus.
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PMID:Effects of cell cycle position on ionizing radiation mutagenesis. I. Quantitative assays of two genetic loci in a human lymphoblastoid cell line. 889 75

Previous experiments in our research group showed that 3'-azido-3'-deoxythymidine (AZT) caused increased mutant frequencies (Mfs) at the X-linked hypoxanthine-guanine phosphoribosyltransferase (HPRT) and the autosomal thymidine kinase (TK) genes in human lymphoblastoid cells and that there was a significant positive correlation between AZT incorporation into cellular DNA and AZT-induced TK Mfs. In the current study, the mutagenicity of AZT was further evaluated at the autosomal adenine phosphoribosyltransferase (APRT) gene. AZH1 cells, a human lymphoblastoid cell line heterozygous at the APRT locus, were exposed to 300 microM AZT for 0, 1, 3 or 6 days or to 0, 33, 100, 300 or 900 microM AZT for 3 days (n = 5 flasks/group). A cell cloning assay was used to quantitate APRT Mfs. AZT-induced APRT Mf increased with extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.022, coefficient = 0.93) between AZT incorporation into DNA and AZT-induced APRT Mfs. RFLP analyses indicated that AZT exclusively induced loss of heterozygosity in APRT mutants. These results, which are consistent with findings on the mutagenicity of AZT at the HPRT and TK genes, indicate the need for further investigations on the potential long-term side effects of AZT on humans, especially those who receive AZT for a prophylactic reason.
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PMID:Mutagenicity and loss of heterozygosity at the APRT locus in human lymphoblastoid cells exposed to 3'-azido-3'-deoxythymidine. 1097 Apr 46

7,12-Dimethylbenz[a]anthracene (DMBA) is a rodent carcinogen and a potent in vivo mutagen for the X-linked hypoxanthine guanine phosphoribosyl transferase (hprt) gene of rats and for the lacI transgene of Big Blue mice and rats. Although DMBA is also a powerful clastogen, molecular analysis of these DMBA-induced hprt and lacI mutations indicates that most are single base-pair (bp) substitutions and 1- to 3-bp frameshifts. In the present study, we evaluated the types of mutations induced by DMBA in the autosomal thymidine kinase (Tk) gene of Tk(+/-) mice. Male and female 5- to 6-week-old animals were injected i.p. with DMBA at a dose of 30 mg/kg. Five weeks after the treatment, hprt and Tk mutant frequencies were determined using a limiting dilution clonal assay in 96-well plates. We established conditions for the automated identification of wells containing expanded lymphocyte clones using the fluorescent indicator alamarBlue. This procedure allowed the unbiased identification of viable clones and calculation of mutant frequencies. In male mice, DMBA treatment increased the frequency of hprt mutants from 1.8 +/- 1.1 to 34 +/- 9 x 10(-6), and Tk mutants from 33 +/- 12 to 78 +/- 26 x 10(-6); treated female mice had a significant but lower increase in hprt mutant frequency than did males. Molecular analysis of DMBA-induced Tk mutants revealed that at least 75% had the entire wild-type Tk allele missing. The results indicate that the predominant types of DMBA-induced mutation detected by the autosomal Tk gene are different from those detected by the X-linked hprt gene. The Tk gene mainly detects loss of heterozygosity mutation, whereas the majority of mutations previously found in the hprt gene were point mutations.
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PMID:7,12-dimethylbenz[a]anthracene-induced mutation in the Tk gene of Tk(+/-) mice: automated scoring of lymphocyte clones using a fluorescent viability indicator. 1115 61


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