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)

We have established a system for the study of DNA-sequence specificity at a functionally heterozygous thymidine kinase (tk) locus in a human lymphoblastoid cell line (TK6). Characterization of the parental locus demonstrated that the 2 tk alleles were fortuitously distinguished by differential gene expression. One round of PCR amplification yielded a specific tk cDNA product only for the functional parental allele. Analysis of cDNA from newly mutated alleles which retain substantial levels of expression is thus simplified. Amplification and sequencing of tk genomic sequences was used for analysis of low expression mutants, and in order to distinguish and characterize deletion and splicing mutations. DNA-sequence analysis of the parental locus identified a frameshift in tk exon 4 of the non-functional parental allele, and surprisingly, an exon 7 frameshift mutation in the functional tk allele. This exon 7 frameshift results in a predicted alteration of the final 21 amino acids of the TK protein, and a C-terminal extension of 131 additional amino acids. Since TK6 is phenotypically TK+, we can infer that this major C-terminal modification does not eliminate enzymatic activity. The system was utilized for the analysis of 36 spontaneous TK- mutants. Loss of heterozygosity accounted for 58% of the mutations, 11% were attributable to intragenic deletions, and the remainder involved point mutations, primarily G:C to A:T transitions.
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PMID:DNA-sequence specificity of mutations at the human thymidine kinase locus. 769 Aug 92

The objective of this work is to identify a heteromorphism within the thymidine kinase (Tk1) gene which can be used to assay for allele loss by means of PCR. Intron F of mouse Tk1 contains two (CA)n microsatellite sequences separated by 107 bp of non-repetitive sequence. We tested this region for heteromorphism in L5178Y mouse lymphoma cells. A PCR primer pair designated Agl1 yielded products of 396 and 194 bp from L5178Y tk+/- genomic DNA. The 194-bp product resulted from a secondary binding site between the two (CA)n repeats for the forward Ag11 primer and was not produced from tk-/- mutants that had lost the functional Tk1b allele. Agl2 primers produced two PCR products of 523 and approximately 440 bp and Agl3 primers produced products of 579 and approximately 500 bp. In both these cases, the difference in product size was approximately equal, indicating that Intron F is approximately 80 bp shorter in the non-functional Tk1a allele than in Tk1b. This heteromorphism forms the basis for an assay for allele loss by means of PCR. Agl1 and Agl3 primers yielded additional products of 91 and 274 bp, respectively, consistent with sizes expected from the mouse Tk1 pseudogenes (Tk1-ps). Our conclusions drawn from an analysis of 122 mutants for Tk1b loss using Agl2 primers agreed with previous analysis of the NcoI heteromorphism. Thus, a simple PCR-based analysis can identify Tk1b loss in the L5178Y mouse lymphoma cells.
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PMID:Identification of a heteromorphic microsatellite within the thymidine kinase gene in L5178Y mouse lymphoma cells. 900 28

The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.
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PMID:Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses. 1895 77

We exploited the differential activation of hypoxia-inducible factor (HIF)-dependent gene expression in tumors versus normal tissue for the design of a targeted oncolytic herpes simplex virus type-1 (HSV-1). A gene that is essential for viral replication, infected cell polypeptide 4 (ICP4), was placed under the regulation of an HIF-responsive promoter and then introduced into the thymidine kinase locus (U(L)23) of HSV d120, which contains partial deletions in the two endogenous ICP4 genes. Recombinant HIF-HSV was isolated and their derivation from d120 was verified by expression of a truncated, non-functional form of ICP4 protein. Disruption of the U(L)23 locus was confirmed by loss of thymidine kinase expression and resistance to acyclovir. Unexpectedly, HIF-HSV expressed ICP4 and induced tumor cell lysis at similar levels under normoxia and hypoxia. The lack of HIF-dependent ICP4 transgene expression by HIF-HSV was due to two factors that have not previously been reported-reversion of the ICP4 gene region to its wild-type configuration and increased HIF-transcriptional activity under normoxia when cells were infected with any strain of HSV-1. The findings that an oncolytic HSV-1 is genetically unstable and can activate a tumor-related promoter in a non-specific manner have important implications for any proposed use of this virus in cancer therapy.
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PMID:Development of an oncolytic herpes simplex virus using a tumor-specific HIF-responsive promoter. 2093 Aug 60

Herpes simplex virus (HSV) infections can be treated efficiently by the application of antiviral compounds. Several drugs on the basis of acyclovir (ACV), penciclovir (PCV) and foscarnet (FOS) have been approved. Resistant viral isolates can be observed especially in immunocompromised patients, who are treated with antivirals for long time intervals. That is why methods for analysis of HSV resistance to antiviral drugs have to be available in virological laboratories. We analyzed HSV type 1 (HSV-1) and type 2 (HSV-2) isolates resistant to ACV for correlation between phenotypic and genotypic drug resistance using tetrazolium reduction assay as well as sequencing of thymidine kinase (TK) and DNA polymerase (pol) genes. All strains were characterized as cross-resistant to PCV, brivudin and susceptible to cidofovir. In addition, three sequential isolates were resistant to FOS. Genotypic analysis revealed high polymorphism of TK among HSV-1 isolates and high polymorphism of DNA pol among both HSV-1- and 2 isolates. In nearly half of ACV-resistant strains, nucleotide insertions and deletions, responsible for a frameshift and the synthesis of a non-functional TK could be related to resistance. In the remaining strains, there were non-synonymous nucleotide substitutions which were not known as part of gene polymorphism. In conclusion, for reliable interpretation of genotypic resistance, a database of non-synonymous mutations has to be established.
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PMID:Testing of herpes simplex virus for resistance to antiviral drugs. 2117 3

We investigated the homology dependency of recombination in thymidine kinase (tk)-deficient mouse fibroblasts. Cells were transfected with DNA constructs harboring a herpes tk gene (the "recipient") rendered non-functional by an oligonucleotide containing the recognition site for endonuclease I-SceI. Constructs also contained a "donor" tk sequence that could restore function to the recipient gene through spontaneous gene conversion or via repair of a double-strand break (DSB) at the I-SceI site. Recombination events were recoverable by selection for tk-positive clones. Three different donors were used containing 16, 25, or 33 mismatches relative to the recipient. The mismatches were clustered, forming an interval of "homeology" relative to the recipient sequences. We show that when homeologous sequences were surrounded by high homology, mismatches were frequently included in gene conversion events. Notably, conversion tracts from spontaneous recombination included either all or none of the mismatches, suggesting that recombination must begin and end in high homology. This requirement was relaxed for events that occurred near an induced DSB, as a significant number of these latter conversion tracts had one end positioned within homeology. Knock-down of mismatch repair showed that incorporation of mismatches into gene conversion tracts can involve repair of mismatched heteroduplex intermediates, indicating that mismatch repair does not necessarily impede homeologous genetic exchange. Our results illustrate (1) genetic exchange between homeologous sequences in a mammalian genome is enabled by nearby homology, (2) proximity to a DSB impacts the homology requirements for where genetic exchange may begin and end, and (3) mismatch correction and previously documented anti-recombination activity are separable functions of the mismatch repair machinery in mammalian cells.
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PMID:Mismatch tolerance during homologous recombination in mammalian cells. 3010 93