Thymidine Kinase Activity of Herpes Simplex Virus Temperature-Sensitive Mutants
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Abstract:
Eighteen temperature-sensitive (ts) mutants of herpes simplex virus type 1 were studied with regard to their ability to synthesize virus-induced thymidine kinase (TK) at permissive (34°) and nonpermissive (39°) temperatures. Five mutants were TK– at both temperatures (TK– mutants), 10 were TK+ at both temperatures (TK+ mutants) and 3 showed reduced TK activity at 39° but not at 34° (TK± mutants). Two of the 3 TK± mutants were ts in the production of TK at 39° while the third mutant synthesized a ts TK at both temperatures. Polyacrylamide gel electrophoresis of extracts of both uninfected and TK-mutant-infected cells exhibited one major and one minor peak of TK activity, whereas extracts of wild-type (WT) virus-infected cells revealed at least one additional major peak of TK activity. Gel patterns of TK± mutants were similar to those of the WT virus in 34 and 39° extracts; however, a reduction in TK activity of the viral peaks was observed in 39° extracts.Keywords:
Thymidine kinase
Thymidine
Wild type
We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine >> L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs.
Pseudorabies
Thymidine kinase
Stereospecificity
Thymidine
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Thymidine
Thymidine kinase
Deoxyuridine
clone (Java method)
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Thymidine
Thymidine kinase
Hymenolepis diminuta
Nucleotide salvage
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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA reappraisal of the effect upon thymidine kinase of thymidine derivatives carrying large groups at the 5'-positionS. Elaine Barrie, Lawrence C. Davies, John A. Stock, and Kenneth R. HarrapCite this: J. Med. Chem. 1984, 27, 8, 1044–1047Publication Date (Print):August 1, 1984Publication History Published online1 May 2002Published inissue 1 August 1984https://pubs.acs.org/doi/10.1021/jm00374a018https://doi.org/10.1021/jm00374a018research-articleACS PublicationsRequest reuse permissionsArticle Views53Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.
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Thymidine kinase
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Brugia pahangi
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