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)

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

Plasmids that replicate autonomously in mouse L cells were constructed by inserting random genomic DNA fragments from Ltk- cells into a plasmid containing the HSV-1 thymidine kinase gene with a truncated low-efficiency promoter. HAT resistance was used as a selective marker. The presence of free plasmids in the DNA of transformants was demonstrated by hybridization with a specific plasmid probe, by electron microscopic visualization of circular DNA, and by recovering these plasmids by E. coli transformation. Nineteen different DNA fragments were isolated. They were characterized as murine autonomously replicating sequences by Mbol restriction endonuclease sensitivity, by bromodeoxyuridine substitution, by copy number determination, and by segregation analysis. Sequence analysis of the inserts of nine plasmids revealed a conserved element of 12 bp (CTCATGAGAGGCCAA) in five out of nine autonomously replicating sequences.
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PMID:Murine genomic DNA sequences replicating autonomously in mouse L cells. 291 71

The induction of class II antigen on primary macrophages and on several macrophage cell lines has been demonstrated after exposure to IFN-gamma. The murine macrophage cell line PU5 (H-2d haplotype) does not express class II antigen constitutively and only trace levels are detected after induction with IFN-gamma. In an attempt to understand the nature of the defect in PU5, stable macrophage hybrids were derived by fusing a thymidine kinase-negative variant of PU5 with thioglycollate elicited peritoneal macrophages from CBA/J mice (H-2k haplotype). Hybrid clones isolated after HAT selection had approximately 35% more DNA than the PU5 parent suggesting that chromosomal loss had occurred post-fusion. While none of the hybrids expressed class II antigens of the k haplotype, constitutive expression of class II antigens of the d haplotype was detected by both micro-ELISA and by flow cytometry. Incubation of these hybrids with IFN-gamma resulted in a further increase in class II expression. Co-ordinate reactivation of both I-A and I-E antigens was observed in the PU5-macrophage hybrids. In contrast, although the IFN-gamma receptor on PU5 and the macrophage hybrids was indistinguishable by Scatchard analysis, neither intracellular class II antigen nor transcription of class II mRNA was detected in IFN-gamma-treated PU5. Reactivation was dependent on the PU5 fusion partner and was not related to a general post-fusion event as hybrids formed between PU5 and the murine fibroblast line A9 did not express class II antigen constitutively or after IFN-gamma induction.
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PMID:Reactivation of class II antigen expression in murine macrophage hybrids. 296 70

A characteristics is given of clone A238 of the Chinese hamster cells deficient in thymidine kinase (TK). The isolation procedure is described. Upon transformation with the aid of DNA of plasmids, containing thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1) clone A238 cells show frequency (7.10(-5) and efficiency (130 TK+ colonies per 1 microgram of plasmid DNA) compatible with those of mouse line LMtk- cells. Modified transformation and selection conditions of clone A238 cells expressing TK-gene of HSV1 are demonstrated. A simple method is described for discriminating somatic cells, expressing either their proper or a virus TK-gene according to the cloning efficiency of cells on the HAT medium containing thymidine in concentration 100 micrograms/ml. It is shown that at the fixed total DNA concentrations a complete replacement of the eukaryotic carrier DNA for the plasmid DNA, containing no tg gene of HSV1, decreases but only insignificantly the frequency and efficiency of transformation.
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PMID:[Genetic transformation of somatic cells. I. Clone of Chinese hamster cells defective in thymidine kinase and characterized by high transformation efficiency]. 298 62

Subclones were isolated both on selective and nonselective medium from the Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV-1) and varying in the rate of the loss of transformant phenotype. The study of the stability of thymidine kinase-positive (TK+) phenotype in cell populations the subclones shows that the nonstability and the rate of the loss of transformant phenotype are the characters that are inherited in the cell generations. Durable cultivation on a HAT-selective medium may lead to a complete or partial (expressed as a reduced rate of the loss of the character) stabilization of TK+-phenotype of the cells of transformant clones. The rate of stabilization of TK+-phenotype may differ depending on the structure of transforming DNA introduced into cells of transformant clones.
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PMID:[Genetic transformation of somatic cells. V. Inheritability of the rate of loss of the trait and the stabilization of the transformant phenotype]. 299 36

The enhancement effects of ionizing and ultraviolet radiation on the efficiency of DNA-mediated gene transfer were studied. The established cell line, Rat-2, consists of cells that are density-dependent contact-inhibited and produce flat monolayers in vitro. When these cells are infected with SV40 virus, a small fraction of cells becomes morphologically "transformed" due to the stable expression of the viral A-gene. Rat-2 cells are competent for DNA-mediated gene transfer, deficient in thymidine kinase activity (TK-), and will die in HAT selective media. Confluent Rat-2 cells were transfected with purified SV40 viral DNA (via calcium phosphate precipitation), irradiated with either X rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A-gene transformants/survivor compared to unirradiated transfected cells. These enhancements were nonlinear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X rays or 330 MeV/amu argon particles at the Berkeley BEVALAC showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. In both cases the enhancement contained a linear and a higher order component in dose, but the argon ions were at least twice more efficient than X rays in producing enhancement per unit dose. Rat-2 cells transfected with pOT-TK5, X-irradiated, and assayed for either TK transformation or A-gene transformation showed the same dose dependence for radiation enhancement. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK transformation by both X rays and ultraviolet radiation. SV40 A-gene products are not necessary for the radiation enhancement of the efficiency of gene transfer. This in vitro system will be used to study the lesions produced by ionizing radiation on mammalian cell DNA that may act as substrates for integration of exogenously introduced plasmid DNA.
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PMID:DNA-mediated gene transfer efficiency is enhanced by ionizing and ultraviolet irradiation of rodent cells in vitro. I. Kinetics of enhancement. 300 17

A series of stable mutants bearing nuclear genetic markers were developed from the established chicken cell line DU24. The mutants were obtained after mutagenesis of DU24 cells with ethyl methanesulfonate (EMS) or arose spontaneously when plated in the appropriate selective medium. Clones resistant to 5-bromodeoxyuridine (BrdU) were obtained following a two-step selection procedure and analyzed. The BrdUr cells were found to be deficient in thymidine kinase activity and were HAT sensitive. Molecular characterization of these mutants revealed no deletions or other rearrangements, but methylation of some cytosine residues was decreased in the mutants. A similar restriction profile was seen in a series of mutants made resistant to BrdU after cultivation of DU24 cells in increasing concentrations of the drug over a period of six months. Selection of EMS-treated BrdUr cells in 10 microM ouabain gave rise to a clone resistant to both drugs and which was still HAT sensitive. Clones resistant to 6-thioguanine were also isolated, but showed wild-type hypoxanthine phosphoribosyltransferase activity and were HAT resistant. A number of the cell lines isolated were found to be suitable for fusion experiments with both chicken cells and cells from other vertebrate species.
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PMID:Development and characterization of mutant chicken cell lines for somatic cell genetics studies. 316 27

To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a "supersuppressor" selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HATr) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT- mutants - selected by their resistance to 6-thioguanine (TGr) - and TK- mutants - selected by their resistance to 5-bromodeoxyuridine (BrdUrdr) - are HATs. Therefore, from HPRT- TK- double nonsense mutants, whose phenotype would be TGr BrdUrdr (HATs), simultaneous HPRT+ TK+ double phenotypic revertants could be obtained by selecting HATr (TGs BrdUrds) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TGr variants, 53 of which were HATs and the rest HATr. Among 36 TGr (HATs) variants tested, 23 did not revert to HATr, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT- nonsense mutants because they were very stringent (had less than 2% of wild-type [3H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrdr) in 7 of these strains allowed us to isolate 29 TGr BrdUrdr (HATs) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HATr (TGs BrdUrds) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.
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PMID:Isolation and characterization of mammalian cell lines carrying suppressible mutations. 322 37

The thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) was identified by biochemical transformation of 3T3 TK negative (TK-) to 3T3 TK positive (TK+) cells using specific viral DNA sequences. DNA fragments of the viral genome used in this study were obtained from a defined gene library of FLDV genome containing the complete viral DNA sequences. The selection of the converted cells was carried out under the condition of the HAT selection procedure. The results of these experiments revealed that the EcoRI FLDV DNA fragment C (11.2 kbp; 0.611 to 0.718 map units) is able to transform 3T3 TK- to 3T3 TK+ cells. Additional experiments using the subclones of EcoRI DNA fragment C revealed that DNA sequences of 4.1 kbp size between the coordinates 0.669 to 0.718 of the FLDV genome possessed the ability for biochemical transformation, indicating that the TK gene locus is located in this particular region.
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PMID:Identification, mapping and cloning of the thymidine kinase gene of fish lymphocystis disease virus. 334 Nov 49

A spontaneously arising clone, stably resistant to the toxic effects of the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine (hmdU), was isolated from unmutagenized V79.5 Chinese hamster fibroblast cells by a single-step selection procedure. The hmdUr cells were selected in the continuous presence of 30 microM hmdU, a concentration which reduces the plating efficiency of wild-type cells to less than 1% after a 24-h exposure. A line of human HeLa cells were found to be intrinsically resistant to concentrations of hmdU as high as 100 microM. All of the hmdUr cells were found to grow normally in HAT medium, which requires the expression of thymidine kinase activity; be sensitive to the toxic effects of high concentrations of 5-bromo-2'-deoxyuridine, another thymidine analog; have unaltered hmdU nucleotide metabolism, as measured by HPLC analysis of acid-soluble cell extracts; and have decreased levels of hmdU incorporation into DNA. Although high concentrations of 5-hydroxymethyluracil (hmUra) were found to be nontoxic for both wild-type and hmdUr cells, the resistance phenotype could be suppressed by exposing the cells to hmdU and high concentrations of hmUra simultaneously.
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PMID:Resistance to toxic effects of 5-hydroxymethyl-2'-deoxyuridine in mammalian cells. 347 Sep 50


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