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)

Interspecific cell hybrids were constructed by fusion of an antimycin-resistant, thymidine kinase- (TK-) Chinese hamster cell line with a chloramphenicol-resistant, hypoxanthine-guanine phosphoribosyl transferase- (HPRT-) mouse cell line. Hybrids were selected in HAT medium alone, or HAT supplemented with chloramphenicol, antimycin, or both antibiotics. Analysis of the mitochondrial DNA (mtDNA) of these hybrids indicates that antibiotic selection directed at the mitochondrial populations results in retention of the resistant parental genome and loss of the sensitive parental genome.
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PMID:Retention of mitochondrial DNA species in somatic cell hybrids using antibiotic selection. 632 97

Electric impulses (8 kV/cm, 5 microseconds) were found to increase greatly the uptake of DNA into cells. When linear or circular plasmid DNA containing the herpes simplex thymidine kinase (TK) gene is added to a suspension of mouse L cells deficient in the TK gene and the cells are then exposed to electric fields, stable transformants are formed that survive in the HAT selection medium. At 20 degrees C after the application of three successive electric impulses followed by 10 min to allow DNA entry there result 95 (+/- 3) transformants per 10(6) cells and per 1.2 micrograms DNA. Compared with biochemical techniques, the electric field method of gene transfer is very simple, easily applicable, and very efficient. Because the mechanism of DNA transport through cell membranes is not known, a simple physical model for the enhanced DNA penetration into cells in high electric fields is proposed. According to this ' electroporation model' the interaction of the external electric field with the lipid dipoles of a pore configuration induces and stabilizes the permeation sites and thus enhances cross membrane transport.
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PMID:Gene transfer into mouse lyoma cells by electroporation in high electric fields. 632 8

Eucaryotic expression vectors containing two selective markers, the herpes simplex 1 thymidine kinase gene (tk) and the Escherichia coli gpt gene (Eco gpt) coding for a xanthine-guanine phosphoribosyl-transferase ( XGPRT ) were constructed. These plasmids were used to transfect mouse Ltk- cells followed by selection of either tk+ or XGPRT + colonies. The transcription and maturation of the Eco gpt mRNA is dependent on the presence of a eucaryotic promoter sequence at its 5' end and on the presence of a viral intron and a poly(A) addition site at its 3' end ( Mulligan and Berg, 1980). Here, we report that both simian virus 40 (SV40) and polyoma early and late promoters permit the transcription of this gene integrated into the cellular genome. Polyoma DNA fragments lacking the TATA box, or both the TATA and CAAT boxes directing early transcription, efficiently promote Eco gpt expression. Furthermore, a fragment terminating approximately 300 nucleotides upstream of the initiation site of early RNA permits Eco gpt synthesis when the early strand is joined to the Eco gpt-coding strand. The SV40 early promoter is 2- to 3-fold more efficient than the controlling sequence of late transcription. These results strongly suggest that the switch from a predominance of early RNA to that of late RNA occurring after the onset of DNA replication is caused by the increase in the abundance of template and by the concomitant repression of early transcription by the T antigen. The presence of the tk gene in all the plasmids constructed permits the analysis of Eco gpt expression as a non-selected marker in tk+ clones selected for growth in HAT medium.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Both early and late control sequences of SV40 and polyoma promote transcription of Escherichia coli gpt gene in transfected cells. 632 14

Genes coding for the heavy chain of the class I antigens HLA-A2 or HLA-B7 of the human major histocompatibility complex have been introduced into mouse LtK- cells by cotransfection with the herpes simplex virus thymidine kinase gene. HAT-resistant colonies were isolated expressing either HLA-A2 or HLA-B7 as monitored by indirect immunofluorescence. Immunoprecipitation analysis of both antigens by either sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing (IEF) showed that they were identical to the HLA-A2 and HLA-B7 expressed in the human lymphoblastoid cell line JY (homozygous HLA-A2, HLA-B7). However, human cytotoxic T lymphocytes (CTL) generated against JY and CTL clones specific for HLA-A2 or HLA-B7 were unable to recognize the transfectants as targets. These results indicate that the human HLA-A2 (or B7) complexed with the murine beta 2-microglobulin could be an inappropriate target structure for the CTL. However, because the transfectants are not killed by human CTL even in the presence of lectins, it is suggested that other molecules that are not able to overcome the human-mouse species barrier may be involved in the killing mechanism.
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PMID:Expression of the major histocompatibility antigens HLA-A2 and HLA-B7 by DNA-mediated gene transfer. 635 10

We have confirmed the localization of human acid alpha-glucosidase (GAA) to 17q21----q25 and of adenosine deaminase (ADA) to 20q13----20qter by examination of hybrid clones derived from a fusion between a human cell line carrying a 17/20 balanced translocation (17pter----17q25::20q13----20qter;20pter-- --20q13::17q25----17qter) and a mouse line deficient in thymidine kinase. These hybrids were constantly maintained in HAT selective media in order to select for the presence of the human thymidine kinase gene on the intact chromosome 17 (17q21----22) or the 17/20 (17pter----17q25::20q13----20qter) translocation chromosome. We detected human GAA by rocket immunoelectrophoresis, using a heterologous antibody raised against human acid alpha-glucosidase. A clone which contained the 17/20 translocation and no intact chromosome 17 was still positive for GAA. This finding confirms the exclusion of GAA from 17q25----17qter reported by Nickel et al. (1982). Combined with earlier results (Weil et al. 1979), GAA can be assigned to 17q21----17q25. A clone which contained only the 17/20 translocation chromosome and no intact chromosome 20 contained ADA. This confirms the previous localization of ADA to 20q13.2----qter by gene dosage studies (Philip et al. 1980).
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PMID:Confirmation of the regional localization of the genes for human acid alpha-glucosidase (GAA) and adenosine deaminase (ADA) by somatic cell hybridization. 637 91

Linear hepatitis B virus (HBV) DNA, excised from a recombinant plasmid with EcoR1, was purified by preparative electrophoresis on agarose gels and incubated with phage T4 ligase to form either monomeric or dimeric closed circles. Thymidine kinase deficient mouse L cells were cotransfected with thymidine kinase (tk) and circular HBV DNAs and grown in hypoxanthine medium. Colonies of tk-transformed cells, selected after 3-4 weeks of incubation and subcultured in HAT medium, synthesized either hepatitis B surface antigen (HBsAg) alone or HBsAg in combination with hepatitis B e antigen (HBeAg). The various cell colonies differed in plating efficiency, growth rates, cellular appearance, and extent of viral antigen synthesis. Southern hybridization analysis showed the presence of HBV-related sequences in high molecular weight DNA prepared from cells expressing viral antigens. Digestion of cellular DNAs with restriction endonucleases indicated integration of the entire viral genome.
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PMID:The hepatitis B virus as a molecular model for chronic infection: synthesis of hepatitis B surface and e antigens in mouse L cells transfected with closed circular viral DNA. 639 45

In order to facilitate cloning of genes for cell surface molecules, we cotransfected LTK- mouse fibroblasts with thymidine kinase (TK) genes and total human or mouse DNA. TK+ cells, selected by growth in HAT medium, were stained with fluorochrome conjugated monoclonal antibodies or other fluorescent ligands which bind to one or another membrane differentiation antigen or receptor. We isolated fluorescent transfectants expressing these molecules using a fluorescence activated cell sorter (FACS). For some antigens, spontaneous gene amplification occurred. By repeated cycles of FACS sorting and regrowth we obtained high expressing clones. We then isolated cDNA and genomic clones using selected cDNA probes to screen phage with cDNA inserts. DNA from virtually any tissue source transfected equally well for the various molecules except for DNA from a trophoblast derived choriocarcinoma cell line which did not transfect for Leu-2.
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PMID:Transfection and cloning of genes for membrane antigens using the FACS. 644 77

Chinese hamster ovary (CHO) cell lines heterozygous at both the adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci were used for single-step selection of spontaneous and induced mutants resistant to 8-azaadenine (AAr), 6-thioguanine (TGr), ouabain (OUAR), or 5-fluorodeoxyuridine (FUdRr). Mutation data are reported for direct mutagens (EMS, ethyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NQO, 4-nitroquinoline 1-oxide) and promutagens (DMN, dimethylnitrosamine; BP, benzo[a]-pyrene) activated by rat-liver homogenates. Optimal plating densities were established for AAr, TGr, OUAR and FUdRr. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP were 2--4 d for AAr, 6--8 d for TGr, 3 d for OUAR, and 1--3 d for FUdRr. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP showed locus-specific differences in sensitivity. Of 61 clonal isolates resistant to AA and assayed for APRT activity, 87% had less than or equal to 5% wild-type activity; of 30 TGr clones assayed, 83% had less than or equal to 5% wild-type HGPRT activity. Of 42 FUdRr clones assayed, 98% had less than or equal to 1% wild-type TK activity. 50 clones selected in medium containing FUdR displayed cross-resistance to 5-bromodeoxyuridine (BUdR) and trifluorothymidine (TFT) and all were sensitive to HAT (hypoxanthine--amethopterin--thymidine) medium. The tk locus showed the largest mutational response as a function of cell survival after mutagen treatment. The rapid expression kinetics for FUdRr and the possibility that the locus detects a broader spectrum of genetic lesions than the other drug-resistance markers are discussed in terms of a sensitive screening assay for detecting potential mutagens.
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PMID:Mutagenicity testing in mammalian cells. II. Validation of multiple drug-resistance markers having practical application for screening potential mutagens. 644 64

A cell line from the Walker carcinosarcoma 256 of the rat has been established in suspension culture in medium with 5% bovine calf serum for over 350 generations, with an average population doubling time of 17 h, a plating efficiency of 56%, a colony forming efficiency of 32%, and a good capacity to form colonies in soft agar. The cells are morphologically indistinguishable from those in the solid tumor and ascites as checked by transmission and scanning electron microscopy. The karyotype is characterized by a modal number of 65 chromosomes and by the presence of a marker metacentric chromosome. The cells express thymidine kinase, gamma-glutamyl transpeptidase, and alkaline phosphatase; are agglutinable by concanavalin A; and can be synchronized by the triple thymidine block. They induce primary tumors, both subcutaneously (solid) and intraperitoneally (ascitic), in the rat; are able to metastasize upon injection by the tail vein; and invade the chorioallantoic membrane of the chick embryo. Cells in suspension can be transferred to monolayers, considerably decreasing their tumorigenicity without affecting the other parameters studied, and can be switched back to suspension culture. DNA-mediated transfection showed that DNA from these cells can transform the NIH-3T3 line. Upon growth of the monolayers in a BrdUr-containing medium, a sub-line was established that was cloned into a thymidine kinase-deficient line unable to grow in HAT medium and with properties otherwise similar to those of the parental wild type cells.
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PMID:Establishment and characterization of cell lines from the Walker carcinoma 256 able to grow in suspension culture and deficient in thymidine kinase. 646 74

We have studied multiple step bromodeoxyuridine (BrdU) resistance in Friend leukemia cells. The mutation rate to 30 micrograms/ml resistance was 5.1 X 10(-5) per cell per generation, and to 100 micrograms/ml was 3.7 X 10(-7) per cell per generation. Resistant variants could not be obtained in a single step using BrdU concentrations higher than 100 micrograms/ml. Three clones isolated through multiple step selection were resistant to 640 micrograms/ml of BrdU and deficient in thymidine kinase, although their ability to transport radiolabeled thymidine was unimpaired relative to wild type. All three clones had low reversion frequencies, as judged by plating efficiencies in couterselective HAT medium. Two such revertant clones were isolated and tested for their forward mutation frequency in BrdU. No resistant clones were obtained when as many as 5 X 10(7) cells were tested. This observation argues against the hypothesis that the Friend cells possess two functional thymidine kinase loci and that the revertants represent a heterozygous condition. We conclude that the hypothesis of null mutations within a hemizygous or heterozygous thymidine kinase locus is sufficient to account for high-level BrdU resistance in Friend leukemia cells.
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PMID:Bromodeoxyuridine resistance: thymidine transport and phosphorylation in Friend leukemia cells. 658 6


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