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)

Plasmid pTKx-1, containing the herpes simplex virus gene for thymidine kinase (TK) inserted into the BamHI site of plasmid pBR322, was introduced into Ltk- cells by calcium phosphate precipitation in the absence of carrier DNA. Line 101 is a TK+ derivative of Ltk- that contains multiple copies of pTKx-1 in a multimeric structure. A derivative of 101 that retained but no longer expressed the herpes simplex TK genes (termed 101BU1) and derivatives of line 101BU1 that reexpressed the genes (termed 101H1, 101HC, and 101HG) were selected. The TK genes in 101BU1 were hypermethylated relative to those in the TK+ parent and derivatives. Growth of 101BU1 in the presence of the methylation inhibitor 5-azacytidine resulted in an average 13-fold increase in the number of TK+ reexpressors, DNA from 101BU1 was inactive in secondary gene transfer, whereas DNA from 101 and from TK+ reexpressors was active. These data support a causative relationship between DNA methylation and decreased gene expression. All TK+ reexpressors examined had DNA rearrangements involving TK DNA.
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PMID:Expression of transferred thymidine kinase genes is controlled by methylation. 618 59

beta 2-Microglobulin (beta 2m) is expressed on the cell surface after introduction of a beta 2mb (C57BL/6N) genomic clone into thymidine kinase-deficient mouse L cells by cotransformation using the calcium phosphate precipitate method. Stable transformant cell lines were identified that express the beta 2mb allele, as determined by reaction of the cells with appropriate monoclonal antibodies and by two-dimensional gel electrophoresis of endogenously labeled immunoprecipitates of cell extracts. These beta 2mb transformants now express ly-m11.2, as detected by an indirect radioimmunoassay. A plasmid subclone of the beta 2mb gene that contains an 8.4-kilobase insert, after introduction into mouse L cells, similarly directs the synthesis of both the beta 2mb and the ly-m11.2 antigens. Thus, the beta 2mb and ly-m11.2 determinants most likely represent sites on the same protein structure.
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PMID:Linkage of beta 2-microglobulin and ly-m11 by molecular cloning and DNA-mediated gene transfer. 618 62

The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.
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PMID:Structure and expression of human globin genes introduced into mouse fibroblasts. 627 97

Treatment of Ltk- cells with the calcium antagonists, verapamil and diltiazem, but not nifedipine, causes a 3-fold enhancement of the frequency of transfer of the cloned gene for herpes simplex virus thymidine kinase (HSV-tk). The frequency of phenotypic expression of the HSV-tk DNA was 20 to 34 times higher than that of genotypic transformation. Phenotypic expression was also 2.3 to 2.6 times increased when 20 micrograms/ml of verapamil was present during calcium phosphate-mediated DNA transfection.
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PMID:Verapamil enhances the efficiency of DNA-mediated gene transfer in mammalian cells. 630 63

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] (ACV), a potent antiviral compound, was phosphorylated to the same extent by extracts from untreated and iododeoxyuridine-treated Epstein-Barr virus-containing latent D98/HR-1 somatic hybrid cells. ATP was the preferred phosphate donor over other nucleoside triphosphates. The cytosol extract from D98/HR-1 cells effected optimum phosphorylation of thymidine at pH 8.0, whereas ACV was phosphorylated equally well over a wide pH range. Electrophoretic analysis of thymidine kinase-, deoxycytidine kinase-, and ACV-phosphorylating activities from both untreated and iododeoxyuridine-treated cell extracts displayed identical properties. A small part (5 to 10%) of the loaded ACV-phosphorylating activity seemed to migrate with the deoxycytidine kinase activity from cytosol. dTTP and dCTP, at relatively high concentrations, partially inhibited ACV-phosphorylating activity. The results suggest that Epstein-Barr virus does not code for its own thymidine kinase and that phosphorylation of ACV in Epstein-Barr virus-producing cells is carried out by multiple or as yet unidentified ATP-dependent nonspecific cellular phosphotransferases.
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PMID:Phosphorylation of acyclovir in vitro in activated Burkitt somatic cell hybrids. 631 70

Herpes simplex virus thymidine kinase gene and pBR322 DNA (in large excess to the thymidine kinase gene) were introduced into mouse L cells by calcium phosphate DNA-mediated gene transfer. DNA fragments encompassing six junctions between the exogenous DNAs have been cloned and their nucleotide sequences determined. Analysis of these sequences has shown that stretches of partial homology involving from 20-50 base pairs are present near the points at which joining occurs between the donor molecules. The structure of the junction sequences suggests that the recombination event involves the alignment of the two donor DNA molecules at partially homologous regions followed by staggered cutting and joining. One donor molecule is always cut in the region of partial homology, while the second is cut at some distance that is a small multiple of 13.5 +/- 0.5 base pairs away (at 0, 14, 27, 39, 41, and 54 base pairs). In the three junctions where the second cut is far from the region of homology, a 17- to 19-base-pair segment of DNA separates the donor sequences. In all cases the origin of this "filler" DNA appears to be oligonucleotides derived from pBR322.
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PMID:A pattern of partially homologous recombination in mouse L cells. 632 Jan 65

We have developed a new recombinant DNA cloning system to isolate directly the functional unit of the human thymidine kinase (TK) gene. The system utilizes a cosmid vector that can shuttle cloned DNA sequences between bacteria and mammalian cells. A complete human cosmid library was constructed and DNA from the total library was transfected to mouse L cells deficient in TK (LTK-) by calcium phosphate precipitation. The transfected cells were then selected with hypoxanthine/aminopterin/thymidine (HAT) medium, and one HAT-resistant cell clone was isolated. This cell line became resistant to HAT selection by acquiring the TK gene derived from the human cosmid library. As the cosmid vector contains the cohesive ends of the bacteriophage, we could directly retrieve the human DNA sequences from the transformed mouse L cells. Total DNA from the transformed TK+ L cells was packaged in vitro with lysogenic bacterial extracts and used to infect Escherichia coli. One of the two recombinant cosmids isolated contained a 43.8-kilobase human DNA insert and was capable of converting TK- L cells to the TK+ phenotype in both acute and stable transformation assays. Thus, we have isolated the functional human TK gene in this recombinant cosmid. The gene was further localized on a 14.5-kilobase BamHI DNA fragment, and it transcribed a mature mRNA of about 1,500 nucleotides. This method of gene isolation has several special features: (i) an intact structural gene can be cloned directly based on its function without knowledge of its amino acid or nucleotide sequence; (ii) the functional gene sequences can be recovered faster and more efficiently than with the usual DNA transfection method; and (iii) in conjunction with cell-sorting techniques, this method can be used to clone genes encoding cell surface markers.
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PMID:Direct isolation of the functional human thymidine kinase gene with a cosmid shuttle vector. 632 Jan 87

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.
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PMID:High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts. 632 Nov 61

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.
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PMID:Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome. 632 72

A sensitive enzyme assay utilizing [125I]iododeoxyuridine as the substrate and CTP as the phosphate donor in combination with isozyme-specific antisera was used for direct detection and typing of herpesvirus deoxythymidine kinase (dTk) in clinical specimens. An investigation of 16 coded vesicle fluid specimens, taken in connection with varicella-zoster virus (VZV) and herpes simplex virus infections, revealed viral dTk activity in 14 samples. All positive samples except one were taken within 5 days after the onset of illness. Serological typing of the dTk activities easily established whether the vesicles were caused by VZV, herpes simplex virus type 1, or herpes simplex virus type 2. The results were obtainable within 5 h and were in agreement with the results achieved by immunofluorescence tests or by virus isolation when positive. Acute- and convalescent-phase sera from patients with VZV infections were analyzed with regard to dTk isozyme composition. All sera collected within 5 days after the onset of varicella were found to contain elevated levels of dTk activity. By the use of isozyme-specific antisera and gel electrophoresis, it was possible to show the presence of both cellular and VZV dTk's. Among the 13 acute-phase sera from zoster patients, only 2 were found to be VZV dTk positive. Convalescent sera, in most cases collected 15 days or more after the onset of illness, were also found to be devoid of VZV dTk. The relevance of the results and the possible use of these methods for viral diagnostics are discussed.
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PMID:Rapid diagnosis of varicella-zoster virus infection by detection of viral deoxythymidine kinase in serum and vesicle fluid. 633 48


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