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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)

Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.
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PMID:Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization. 629 Aug 73

Thymidine kinase-deficient Chinese hamster ovary (CHO) cells were genetically transformed with the BamHI restriction fragment encoding the thymidine kinase gene of herpes simplex virus (HSV-tk). We have observed considerable clonal variation among independent CHO sublines with respect to transformation competence for the DNA-mediated gene transfer of HSV-tk. Transformation frequencies greater than or equal to 3 X 10(-4) were observed consistently in one subline, with a transformation efficiency of approximately 1 transformant per ng viral gene. The frequency and efficiency of transformation we observed in this system are at least 10-fold greater than those previously reported for DNA-mediated transformation of CHO cells by HSV-tk. All of the CHO HSV-tk+ transformants examined were stable for the transferred genotype in the absence of selection, and all showed evidence of co-transformation by unselected plasmid pBR322 sequences.
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PMID:DNA-mediated gene transfer in Chinese hamster ovary cells: clonal variation in transfer efficiency. 629 69

Two plasmids containing nonoverlapping deletions of the herpes simplex virus thymidine kinase gene were introduced into thymidine kinase-deficient mouse L cells by DNA-mediated gene transfer. Thymidine kinase-producing transformants were generated by a mixture of the two plasmids at a frequency significantly greater than that generated by either plasmid alone. Southern blot analyses demonstrated that functional thymidine kinase genes were generated by homologous recombination between the two deletion mutants.
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PMID:Recombination during gene transfer into mouse cells can restore the function of deleted genes. 629 29

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.
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PMID:DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA. 629 44

Mouse L cells deficient in thymidine kinase (tk-) were transfected with either monomeric or dimeric circular HBV DNA and co-transformed with tk+ DNA derived from herpes simplex virus. Thymidine kinase transformed cell colonies were harvested and maintained as separate cell lines. Either HBsAg, HBeAg, or HBsAg and HBeAg were detected in culture fluids from the various cell colonies. Immunofluorescent stains of cells from colonies synthesizing HBsAg and/or HBeAg showed that these antigens were present in cytoplasm. The monomeric form of circular HBV DNA was more efficient in producing cells synthesizing large amounts of HBsAg and HBeAg than the dimeric form. The patterns of integration of HBV DNA differed between cells synthesizing HBsAg and those synthesizing HBsAg and HBeAg. This in vitro system for the expression of HBV DNA now allows a detailed study of the synthesis and function of HBeAg and the interaction of HBV DNA and the retroviral DNA present in mouse L cells. Furthermore, it may be possible to develop a hepatitis B virus vaccine based on HBeAg as the viral antigen.
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PMID:Expression of hepatitis B viral antigens in animal cells transfected with viral DNA. 630 78

The structural gene for herpes simplex virus (type 1) thymidine kinase was cloned downstream from the lambda phage high efficiency leftward promotor in a plasmid (pHETK2) also containing the gene for the lambda cI857 temperature-sensitive repressor. Thymidine kinase is synthesized as a run-on product containing the NH2 terminus of the lambda N protein. Heat inactivation of the lambda repressor by growth at 42 degrees C results in the accumulation of thymidine kinase as approximately 4% of the total soluble cellular protein. Thymidine kinase has been purified to greater than 95% homogeneity by high speed centrifugation, ammonium sulfate fractionation, and Sephadex G-100 and hydroxylapatite column chromatography. Thymidine kinase has a subunit Mr = 42,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a dimer during Sephadex G-100 chromatography and glycerol gradient centrifugation. Thymidine kinase is enzymatically active from pH 6 to 10 with maximum activity at pH 8.5. The enzyme is protected from heat inactivation by thymidine and has a half-life at 40 degrees C of 30 min in the presence of thymidine and 3 min in its absence. Thymidine kinase displays Michaelis-Menten kinetics with apparent Michaelis constants of 0.6 and 118 microM for thymidine and ATP, respectively. Iododeoxycytidine is a competitive inhibitor of thymidine with an apparent Ki of 14 microM. The anti-herpes drug acyclovir (9-[(2-hydroxyethoxy)methyl]guanine) also appears to be a competitive inhibitor of thymidine (Ki of approximately 300 microM) but requires 3,000-fold higher concentrations than thymidine to give 50% inhibition. Other nucleoside triphosphates can substitute for ATP in the kinase reaction with the exception of dTTP which appears to inhibit thymidine kinase activity by about 50% when present in concentrations equal to that of thymidine.
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PMID:Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature-sensitive repressor. 631 15

Thymidine kinase in chick embryo retina reaches its highest values on the 8-10th day of development, then declines reaching the lowest value at hatching. The rate of DNA synthesis essentially follows this activity while, in contrast, nucleoside phosphotransferase increases progressively during development. Glucocorticoids at 5 X 10(-6) M lower the level of thymidine kinase in isolated retinas of chick embryo. The most effective steroid was hydrocortisone. The effect was observed in retinas from 8-18-day-old chick embryo and, except on the 8th day, was always of the same magnitude. We suggest that a glucocorticoid can be the natural factor responsible for the marked fall in thymidine kinase during development. Brief periods of exposure to steroids increase nucleoside phosphotransferase activity in isolated chick embryo retinas. When the exposure was longer than 3 h this activity was also clearly decreased. We conclude that other factors are responsible for the natural increment which occurs for this activity during development.
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PMID:The effects of glucocorticoids on thymidine kinase and nucleoside phosphotransferase during development of chicken embryo retina. 631 40

The in vitro replication of eleven different strains of herpes simplex virus type 1 was studied in resident or thioglycollate-stimulated mouse macrophages. The strains of herpes simplex virus differed in the type of cytopathic effect, induction capacity for herpes simplex virus coded thymidine kinase and pathogenicity in the mouse. Herpes simplex virus replicated better in thioglycollate-stimulated macrophages than in resident macrophages. In vitro ageing of macrophages increased their replicative potency. Herpes simplex virus replicated better in macrophages from homozygous bg/bg C57/BL6J mice than in macrophages from their heterozygous littermates. Separation of macrophages on discontinuous Percoll-gradients revealed 4 fractions with identical potency for replication. The ability of herpesvirus to replicate in macrophages varied from strain to strain of virus i.e. Wal greater than Len, clone 4 of Len, greater than L3-2s, JES, Ang-, Ang + path, clone 2 of Len and greater than MDK clones. The ability to cause cytopathology also varied. Only strains Ang- and Ang + path showed limited or late cytopathology in macrophages. The cell-fusing property of herpes simplex virus appeared to be more closely correlated with lower replication rates than production cell rounding. Thymidine kinase- viruses replicated less well than thymidine kinase+ or thymidine kinase(+) strains. Strains of herpes simplex virus with high or low pathogenicity for mice replicated in macrophages to the same degree. The phagocytic activity of macrophages for IgM-coated sheep red blood cells was inhibited earlier by strains of herpes simplex virus of type 2 than by strains of herpes simplex virus of type 1.
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PMID:Replication of HSV-1 in murine peritoneal macrophages: comparison of various virus strains with different properties. 632 Jul 76

Thymidine kinase-deficient syrian hamster cells were cotransfected with recombinant plasmids containing the thymidine kinase (TK) gene of Herpes Simplex Virus Type 1, and either intact or partially deleted SV40 T antigen-coding genes. The transformants were selected by their ability to grow in gHAT medium. After selection and cloning, the TK-positive transformants that also expressed T antigen were tested for the extent of their transformation with respect to a number of characteristics, which included saturation density, ability to grow in soft agar, resistance to butyrate and to dibutyryl-cAMP, and plating efficiency. The combined results of these various tests indicate that cells containing partially deleted SV40 T antigen-coding genes are less transformed than cells containing an intact SV40 T antigen-coding gene. However, the amounts of T antigen are lower in cells transformed by deletion mutants than in cells transformed by wild-type T antigen-coding gene. Our data indicate that both the quantity and the quality of T antigen may be important in determining the degree of transformation in Syrian hamster cells.
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PMID:Transforming potential of deletion mutants of the SV40 T antigen coding gene in Syrian hamster cells. 632 70

Thymidine kinase negative (TK-) Friend cells were transformed with recombinant molecules carrying human globin genes and the thymidine kinase gene of herpes simplex virus type 1 DNA. Transformation frequencies of 1 transformant/microgram donor DNA/1 x 10(6) cells were obtained by standard procedures and this was increased 20- to 30-fold by treating recipient cells with dimethyl sulfoxide or glycerol. Transformed cell lines expressed thymidine kinase activity of viral origin as determined by its insensitivity to 0.2 mM dTTP and electrophoretic mobility in polyacrylamide gels. The physical status of donor DNA in the transformed cells was examined in Hirt precipitates and supernatants by Southern blot hybridization and spot hybridization techniques. This analysis showed that most donor sequences were present in a circular or concatenate configuration, but also was suggestive of some donor sequences being integrated into high molecular weight DNA. Expression of human globin genes and particularly the epsilon-globin gene in the transformed Friend cells was studied by Northern blot hybridization analysis.
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PMID:Transfer of human globin genes to erythroleukemic mouse cells. 632 49


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