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)

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).
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PMID:Vaccinia virus DNA ligase is nonessential for virus replication: recovery of plasmids from virus-infected cells. 198 87

We have used triparental matings to demonstrate transfer (mobilization) of the nonconjugative genetically engineered plasmid pHSV106, which contains the thymidine kinase gene of herpes simplex virus cloned into pBR322, from Escherichia coli HB101 to an environmental isolate of Enterobacter cloacae in sterile drinking water. This is the first demonstration of a two-step mobilization of a genetically engineered plasmid in any type of fresh water, including drinking water. Transfer was mediated by R plasmid R100-1 of E. coli ED2149(R100-1). Matings in drinking water at 15, 25, and 35 degrees C yielded recombinants, the number of which increased with increasing temperature. Numbers of recombinants obtained were 2 orders of magnitude lower than those obtained from matings in Trypticase soy broth. High concentrations of parental organisms (2.6 x 10(8) to 2.0 x 10(9) CFU/ml) were required. During 1 week of incubation in drinking water, number of parental organisms and recombinants resulting from mobilization remained constant in the absence of indigenous organisms and declined in their presence. Using oligonucleotide probes for the cloned foreign DNA (thymidine kinase gene) and plasmid vector DNA (ampicillin resistance gene), we demonstrated that both genes were transferred to E. cloacae in the mobilization process. In one recombinant selected for detailed study, the plasmids containing these genes differed in size from all forms of pHSV106 present in E. coli HB101(pHSV106), indicating that DNA rearrangement had occurred. This recombinant maintained its plasmids in unchanged form for 15 days in drinking water. A second rearrangement occurred during serial passage of this recombinant on selective media.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mobilization of the genetically engineered plasmid pHSV106 from Escherichia coli HB101(pHSV106) to Enterobacter cloacae in drinking water. 203 7

In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase gene (tk), a well regulated viral gene has been chosen for this study. A cosmid library of IBRV has been constructed in Escherichia coli HB101 by cloning partially Sau3A-digested DNA fragments into a cosmid vector, pJB8. Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization. Results showed that this cosmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then used to transform E. coli tdk- mutant strains, Ky895 or C600tdk- for the selection of the IBRV tk gene. The clones able to grow on the selection plates containing 5-fluorouracil, uridine, thymidine and ampicillin were selected and further characterized. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and the sequences complementing the tk activity have been isolated by subcloning. The plasmid, pIBRTK, was shown to grow on selection plates and therefore, retained the ability to complement the tk gene. The E. coli mutant strain C600tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of [3H]thymidine into bacterial DNA over that of C600tdk- mutant.
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PMID:Identification of the thymidine kinase gene of infectious bovine rhinotracheitis virus and its function in Escherichia coli hosts. 302 90

A recombinant plasmid pTKH beta GHp-3 carrying genomic human DNA sequences coding for the beta globin gene covalently linked to the thymidine kinase gene of HSV-1 and the ampicillin resistance region of a bacterial plasmid has been constructed. Using Bal 31 exonuclease, deletion mutants have been obtained from a single HpaI site located 800 bp upstream the 5' end of the beta globin gene and towards the cap site. Recipient mouse erythroleukemic Friend TK- cells were transformed with these molecules. Analysis of donor DNA sequences in transformed cells by Southern blotting and filter hybridization has demonstrated the presence of full length multiple copies. RNA isolated from transformed non-induced Friend cells, carrying one or the other of the donor human beta globin deletion mutants, has been analysed by Northern blot and spot hybridization analyses. Evidence has been obtained which suggests that DNA sequences located upstream the CCAAT box (at -76 bp from the cap site) are required for optimal levels of human beta globin specific RNA in transformed cells.
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PMID:Expression of the human beta globin gene and 5'-deletion mutants in erythroleukemic mouse cells studied by DNA mediated gene transfer. 620 56

Ltk- aprt- mouse L cells were transformed to the tk+ phenotype with 10 ng of the herpes simplex virus-1 thymidine kinase (tk) gene and 20 micrograms of pBR322 or simian virus 40 (SV40) DNA. DNAs from five cloned cell lines show restriction endonuclease fragments that hybridize to both tk and pBR322 or SV40 DNA. In all of the cell lines some of these fragments also contain cellular DNA sequences. The use of carrier DNAs with defined sequences has enabled us to demonstrate that the joining of carrier and selectable gene sequences occurs in mouse cells. In one case we have been able to use the ampicillin resistance marker of pBR322 to "rescue" a recombinant plasmid. An analysis of the junction between pBR322 and tk in this plasmid suggests that a small area of homology (16 of 19 base pairs) might be involved in the recombination process.
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PMID:DNA-mediated gene transfer: recombination between cotransferred DNA sequences and recovery of recombinants in a plasmid. 628 42

Recombinant Charon 4A phages accommodating the Herpes simplex virus (HSV-1) thymidine kinase (tk) gene, the ampicillin-resistance (ApR) gene, and the replication origin of pBR322 were constructed. The phage DNA was introduced into mouse Ltk- cells by a free DNA transfer method or phage-mediated DNA transfer method [Ishiura et al., Mol. Cell. Biol. 2 (1982) 607]. Analyses of the physical state of the transferred DNA in the recipient cell genome showed that a DNA fragment as long as 12.7 kb was integrated intact into 67% and less than 40% of the Ltk- transformant cells by phage-mediated DNA transfer and by free DNA transfer, respectively. We also developed a new rapid method for recovery of the transferred gene from the Ltk+ cell into Escherichia coli; the method depends on the fact that the recombinant lambda phage carrying the ApR gene and replication origin of pBR322 transduces lambda-lysogenic bacteria to ApR and is maintained as a plasmid. Using this method the HSV-1 tk gene from one Ltk+ transformant was rapidly and successfully recovered without any rearrangement of the target sequence.
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PMID:A vehicle for DNA transfer and for recovery of transferred genes: lambda Charon phage-pBR322 hybrid. 631 39

The adeno-associated virus 2 (AAV)-based vector system has been suggested for its potential use in human gene therapy because the wild-type (wt) AAV genome appears to integrate into the human chromosomal DNA in a site-specific manner. We systematically investigated the integration patterns of the recombinant AAV genomes lacking one or both the viral coding sequences. Four recombinant AAV genomes were constructed containing the genes for resistance to tetracycline (TcR) and the herpesvirus thymidine kinase (TK) promoter-driven gene for resistance to neomycin (neoR; vTc.Neo), the genes for resistance to ampicillin (ApR) and TK-neoR (vAp.Neo), the genes for AAV replication (rep) genes and TK-neoR (vRep.Neo), and the AAV capsid (cap) genes and TK-neoR (vCap.Neo). The integration pattern of each of the recombinant AAV genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) analyzed on Southern blots using a neo-specific DNA probe was distinctly different. In addition, in none of the clones examined was the proviral genome covalently linked to the previously described AAV right-junction (Rt.Jn.) human chromosomal DNA fragment, the putative specific-site of integration for the wt AAV genome. Furthermore, whereas a 276-bp DNA fragment could be readily amplified from each of these clones, using a neo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained using the neo- and the Rt.Jn. primer-pair under identical conditions. Fluorescence in situ hybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functional rep gene as determined by rescue of the recombinant AAV genome in the presence of adenovirus. These data suggest that the recombinant AAV genomes integrate at sites that are different from that characterized for the wt AAV genome. These studies may have implications in the development of the AAV-based vector system for its potential use in human gene therapy.
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PMID:Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells. 904 94

The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of two genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase [1]; this generally requires 1-2 months of work for each construct. The single 'knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection (using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knockout vector. This approach leads to inactivation of the gene of interest [2]. Recently, an in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing [3]. An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition. The DNA is then purified, and subsequently electroporated into bacteria. The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events. In the present study, we have modified this system to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs. Our approach led from genomic clone to completed construct ready for transfection in a matter of days. The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs [4-8], simplifying this technically demanding method.
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PMID:Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice. 921 Mar 79

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.
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PMID:A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame. 1072 84

We report the construction of histidine-3 (his-3) strains of Neurospora crassa containing the hygromycin B phosphotransferase gene of Escherichia coli (hph(+)) fused in-frame to the herpes simplex virus thymidine kinase gene (tk(+); Lupton et al. 1991), integrated at the his-3 locus. We also report the construction of two ampicillin-resistant and two kanamycin-resistant his-3 gene-replacement vector plasmids. The combined use of these strains and plasmids for his-3-targeted gene integration allows for the rapid identification of homokaryotic transformants containing the expected gene replacement event.
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PMID:Construction of strains for rapid homokaryon purification after integration of constructs at the histidine-3 ( his-3) locus of Neurospora crassa. 1268 41


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