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)

Direct microinjection of DNA by glass micropipettes was used to introduce the Herpes simplex virus thymidine kinase gene into cultured mammalian cells. When DNA was delivered directly into the nuclei of LMTK-, a mouse cell line deficient in thymidine kinase activity, 50--100% of the cells expressed TK enzymatic activity. In contrast, no TK activity could be detected when the DNA was injected into the cytoplasm. The number of injected LMTK- cells capable of indefinite growth in a TK+ selective medium (that is, transformants) depended on the nature of the plasmid DNA into which the HSV-TK gene was inserted. One cell in 500-1000 cells which received nuclear injections with pBR322/TK DNA gave rise to a viable colony when grown in HAT medium (that is, a TK+ selective medium). The transformation frequency increased to one in five injected cells when specific SV40 DNA sequences were also introduced into the HSV-TK plasmid. With the microinjection procedure transformation frequency was relatively insensitive to DNA concentration and did not depend on co-injecting with a carrier DNA. Most of the transformants were stable in nonselective medium as soon as they could be tested.
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PMID:High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. 625 82

The herpes simplex type 1 biochemically transformed human cell line, HB-1, was fused with thymidine kinase deficient rodent cells, and 18 hybrids were isolated using the HAT-ouabain selection system. The selected enzyme, viral thymidine kinase, was present in all 18 hybrids. In 16 of 18 hybrids the viral gene for thymidine kinase cosegregated with the human gene for adenylate kinase-1 (AK-1). Thirty-six bromodeoxyuridine (BrdUrd) resistant sublines were isolated from the 16 human AK-1 positive hybrids. Each BrdUrd-resistant subline was examined for the presence of the viral TK gene by back-selection in HAT medium, and for human AK-1. In all 36 BrdUrd-resistant sublines the viral TK gene cosegregated with the human AK-1 gene. These results indicate that the transforming viral DNA fragment was associated with a specific human chromosomal region in HB-1 cells.
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PMID:Association of the herpes simplex-1 viral gene for thymidine kinase with the human gene for adenylate kinase-1 in biochemically transformed cells. 626 33

The Epstein Barr nuclear antigen (EBNA) and the rheumatoid arthritis nuclear antigen (RANA) develop in human B lymphocytes that have been infected and transformed by Epstein Barr virus (EBV). Antibodies to RANA and EBNA are found only in individuals with prior exposure to EBV. The purpose of the present studies was to determine the relation of the 2 antigens to each other and to EBV genetic material, in human-rodent somatic cell hybrids. Cultured human B lymphoblastoid cells, Raji, Daudi, and RPMI 4098 were fused with thymidine kinase-deficient mouse or hamster fibroblasts. After selection and cloning in ouabain-HAT medium, the hybrid nature of the surviving cells was confirmed by isozyme analysis. The hybrid clones were analyzed for EBNA by anti-complement im,munofluorescence, and for RANA by anti-immunoglobulin immunofluorescence and immunodiffusion. The results showed that RANA and EBNA segregated entirely independently of each other in the hybrid clones. Two methods were used to detect the presence of EBV DNA sequences in the intracellular DNA of hybrid clones. The 1st method relied on the hybridization of labeled cRNA prepared from virion DNA with DNA from 8 hybrid clones affixed to nitrocellulose filters. The 2nd approach was to hybridize labeled intracellular DNA from 3 hybrid clones to Southern blots of cloned fragments of EBV DNA. These results suggested that the presence of EBV DNA was not sufficient for the expression of either antigen. One stable RANA-positive hybrid cell line contained at least 80% of the EBV genome in the absence of detectable EBNA.
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PMID:Discordant expression of 2 Epstein-Barr virus-associated antigens, EBNA and RANA, in man-rodent somatic cell hybrids. 626 53

Successful shuttling of cloned DNA in eukaryotic cells should allow isolation of expressed genes. We tested the utility of cosmids for moving DNA into and out of eukaryotic cells. The unique cleavage of DNA at the cos site by the terminase function of lambda was exploited to maintain the linkage between the vector and inserted gene sequences, a prerequisite for successful rescue of the transforming DNA from high molecular weight DNA of the eukaryotic transformant. A cosmid recombinant containing the HSV thymidine kinase gene and a lambda recombinant containing the chicken thymidine kinase gene were used to test the feasability of this method. It was found that these recombinants can be rescued with high efficiency from DNA of HAT-resistant cells.
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PMID:Gene shuttling: moving of cloned DNA into and out of eukaryotic cells. 628 Jan 36

We have previously described several series of biochemical transformation experiments in which small defined portions of herpes simplex virus (HSV-1 and HSV-2) DNA encompassing the thymidine kinase (TK) gene were introduced into Ltk- cells by the calcium transfection procedure. The presence of authentic virus TK enzyme in several subcloned cell lines derived from these experiments was confirmed by either the specific incorporation of [125I]iododeoxycytidine into their nuclei or the inhibition of cell growth by the antiviral drug arabinosyl thymine. A panel of 24 independent Ltk+ cell lines receiving either isolated virus DNA fragments or cleaved plasmid DNAs was examined by blot hybridization for both the presence and copy number of virus TK DNA sequences. Most cell lines contained a single virus DNA fragment covalently joined to host (or carrier Ltk-) mouse DNA sequences, but several contained multiple copies of the TK gene. Examination of the structural arrangement of the virus DNA in two early passage multicopy cell lines indicated that the TK gene had integrated into Ltk- cell DNA and then subsequently both viral and flanking cellular sequences were amplified to create up to 20 tandem duplications. In one case, mapping of the adjacent cellular sequences has revealed that the total repeat unit is greater that 23 kilobases (kb) in size. On subsequent passaging, even in HAT medium, the amplified repeat units were not stable and fell to only three to four copies per haploid cell genome. These cell lines should prove useful for additional studies to examine the expression of co-selected non-TK virus sequences and the influence of adjacent cellular DNA sequences on transcription and retransfection of the resident TK gene.
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PMID:Transfection with the isolated herpes simplex virus thymidine kinase genes. II. Evidence for amplification of viral and adjacent cellular DNA sequences. 628 48

We have defined the minimal size and physical map locations in the genomes of both herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) for DNA sequences capable of conferring stable biochemical transformation under thymidine kinase (TK) selection. The experiments involved transfection of Ltk- cells with either isolated virus DNA fragments or cloned pBR322 plasmids containing the 3.5 kilobase (kb) BamHI-O fragment from HSV-1(MP) or the 5.6 kb SalI-G fragment from HSV-2(333). Mapping of restriction enzyme sites within these cloned DNAs, followed by assays for colony formation in HAT medium after transfection with cleaved DNA, localized the biologically active TK-transforming sequences to lie between coordinates 0.300 and 0.313 in HSV-1 and between 0.303 and 0.315 in HSV-2. Experiments with a series of cloned plasmids containing deletions of the BamHI-O fragment towards either the 3'- or 5'-ends of the TK gene indicated that the sequences required for stable HSV-1 TK transformation lay within a 1600 base pair (bp) region at 0.303 to 0.313 map units. An internal deletion mutant plasmid, selected by a novel bacterial transfection assay for the absence of the KpnI site at 0.308, also failed to rescue Ltk- cells. With the exception of cleavage at the StuI site at 0.303 in HSV-2, which reduced activity only eightfold, all cleavages that affected TK transformation reduced the efficiency at least 50-fold. A direct comparison of the HSV-1 and HSV-2 minimal transforming regions with the nucleotide sequence of the structural HSV-1 TK gene indicates that the HSV-2 StuI site lies 30 bp beyond the poly(A) addition site at the 3'-end of TK mRNA. On the other hand, cleavage at the SmaI site in HSV-1 TK, located 80 bp in front of the poly(A) addition point, abolishes colony formation. Comparison of the putative 5'-end of the HSV-2 TK gene defined by transfection assays, with a 250 bp non-transcribed region at the front of the HSV-1 TK gene, suggests that the promoter regions contain a much higher frequency of conserved cleavage sites than do the coding portions of the two genes. Direct nucleotide sequencing of the 5'-flanking sequences for HSV-2 TK confirmed that large portions of the two promoters possess greater than 95% sequence homology. At least 140 bp, but no more than 200 bp, of this 5'-promoter region are essential for efficient transfer and expression of the viral TK gene. Combining the results from HSV-1 and HSV-2, we conclude that a contiguous sequence of 1480 to 1540 bp is necessary to achieve at least 10% of the maximum transformation efficiency.
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PMID:Transfection with the isolated herpes simplex virus thymidine kinase genes. I. Minimal size of the active fragments from HSV-1 and HSV-2. 629 48

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

Genetic variation was studied in several mouse L cell lines containing tandemly repeated herpes simplex virus thymidine kinase (TK) genes introduced by DNA-mediated gene transfer. Variants were obtained after alternate positive and negative selection for TK expression. Three classes of molecular alteration are described. One class consisted of a concerted wave of hypermethylation affecting many sites in all or nearly all of the TK genes. This resulted in genetically stable TK- variants. Of five TK+ transformants from independent transfer experiments, only one, named HM, showed this class of methylation. Hypermethylation was a reproducible phenomenon in HM, yielding TK- variants after selection with either bromodeoxyuridine or acycloguanosine [Acyclovir or 9-(2-hydroxyethy-oxymethyl)guanine]. A second class of alteration consisted of methylation affecting some, but not all, genes in the cluster. This happened in all TK+ (HAT [hypoxanthine-aminopterin-thymidine]-resistant) cell lines investigated, and this second class of methylation was incapable of generating TK- variants. Neither type of methylation was accompanied by genomic rearrangements. The third class of molecular alteration was found among TK+ (HAT-resistant) back revertants of hypermethylated HM TK- derivatives. It consisted of a 10-fold amplification of the hypermethylated TK genes. Demethylation of hypermethylated HM variants was not observed. Thus, hypermethylation in this system can be compensated for by amplification but cannot be reversed.
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PMID:Phenotypic variation associated with molecular alterations at a cluster of thymidine kinase genes. 631 Mar 68

Mouse LTK- cells (H-2k) were transfected with a series of recombinant plasmids consisting of the herpes simplex virus type 1 thymidine kinase (TK) gene linked to fragments of SV40 DNA coding for portions of SV40 T antigen in pBR322, and TK+ transformants (LTK+) were selected in HAT medium. The TK+ transformants were analyzed for SV40 transplantation rejection antigen (TrAg) at the cell surface by reacting them with cytotoxic lymphocytes (CTL) generated to SV40 TrAg in C3H/HeJ (H-2k) mice. The results indicated that the cells transformed by pVBETK-1 and synthesizing full size SV40 large T antigen were efficiently lysed by SV40 CTL. In addition, cells transformed by the plasmid pVBt1TK-1 and synthesizing a truncated 33 K T antigen were also found to be susceptible to lysis by the CTL. However, LTK+ cells that were transformed with the plasmid pVBt2TK-1 and which synthesized a truncated T antigen of 12.3 K did not provide a target for SV40 CTL nor did pVBETK-1-transformed cells that did not express any of the SV40 tumor antigens. Only the pVBETK-1-transformed cells that express 94 K T antigen were able to immunize mice against a challenge of syngeneic SV40-transformed cells. These results suggest that the TrAg expression at the cell membranes of transformed cells may be associated with the proximal half of SV40 T antigen.
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PMID:Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen. 631 Aug 60

We describe the characteristics of a general assay for eukaryote transcription-control sequences using the herpes simplex virus (HSV) thymidine kinase (tk) gene. After transfection of cultured cells with tk-containing recombinant plasmids, two assays were used to measure gene expression: short term or transient levels of tk mRNA and TK enzyme activity, and the rate of biochemical transformation from a TK- to a TK+ phenotype in selective growth medium (HAT). Deletion of the endogenous tk promoter results in 500-fold inactivation of gene expression. Replacement with exogenous transcription-control sequences from the human epsilon globin, mouse beta major globin, simian virus 40 and Moloney murine sarcoma virus (MoMuSV) genomes results in reactivation of gene expression. The presence of enhancers or activators of gene expression can also be conveniently measured. The transient expression assay ranged over two orders of magnitude while the transformation assay was almost two orders of magnitude more sensitive using the same recombinants. Analysis of the transcription-control domains in the MoMuSV LTR sequences shows the presence of both an enhancer and a promoter whose activity equalled that of the tk endogenous promoter. Insertion of the LTR promoter between the LTR enhancer and the tk promoter had little effect on modulating gene expression, suggesting no absolute preference for proximal promoters by this element. The different levels of gene expression obtained appears to be mediated by transcriptional control of full-length tk mRNA. There was an apparent correlation between the results obtained with the transient expression and transformation assays. However, cultured transformed cells all contained roughly the same levels of tk DNA, tk mRNA and tk enzyme activity. We propose that initial expression levels have a major effect in determining the transformation efficiency but that additional genetic controls are superimposed in cells grown in selective HAT medium.
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PMID:Characterization of eukaryotic transcriptional control signals by assay of herpes simplex virus type 1 thymidine kinase. 631 53


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