Thymidine Kinase Isozymes of Normal and Virus-infected Cells

Kit, Saul; Leung, Wai-Choi; Jorgensen, George N.; Trkula, David; Dubbs, D. R.

doi:10.1101/sqb.1974.039.01.084

Cited by 34 publications

(26 citation statements)

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“…The herpes simplex virus type I (HSVI) thymidine kinase (tk) gene is of interest because its expression is regulated during viral growth (1) and because it can serve as a selective marker in the biochemical transformation ofanimal cells with DNA (2, 3). However, apart from its orientation within a restriction fragment of the viral DNA (4), little is known about the basic features of the HSVI tk gene.…”

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Expression of herpes simplex virus type I thymidine kinase gene in Escherichia coli.

Garapin¹,

Colbère-Garapin²,

Cohen-Solal³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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A herpes simplex virus type I DNA fragment containing the sequence coding for thymidine kinase was fused to the very beginning of the Escherichia coli lac Z gene in the three possible reading frames. When the thymidine kinase sequence was in the orientation fit to be transcribed from the lac promoter, functional thymidine kinase was made under lac control in all three cases. Sequences data indicate that translation reinitiation occurs at the 5' end of the thymidine kinase gene after stop signals. Two T+A-rich sequences, which may be part of eukaryotic promoters, are found in the same region.The herpes simplex virus type I (HSVI) thymidine kinase (tk) gene is of interest because its expression is regulated during viral growth (1) and because it can serve as a selective marker in the biochemical transformation ofanimal cells with DNA (2, 3). However, apart from its orientation within a restriction fragment of the viral DNA (4), little is known about the basic features of the HSVI tk gene. We (5) and others (6, 7) have cloned it in Escherichia coli. We then took advantage ofthe availability ofTK-E. coli strains to look for complementation of the bacterial defect by an expressed HSV tk gene. For this purpose, we have characterized the HSVI tk promoter region and fused the gene to a bacterial promoter to force expression. The analysis reported here brings information about eukaryotic gene expression in E. coli as well as about the structure ofthe HSVI tk gene itself. MATERIALS AND METHODSBiohazards. Biohazards associated with the experiments described in this publication have been examined by the French National Control Committee, and the experiments were carried out according to the rules established by the committee.Enzymes and Reagents. Restriction endonucleases Pvu II, EcoRI, and HinclI were purchased from Biolabs, Sst I was from Bethesda Research Laboratories (Rockville, MD), and Bgl II was from Boehringer Mannheim. Reaction conditions were as recommended by the manufacturers. Calf intestine grade I alkaline phosphatase (Boehringer Mannheim) was used as described (5). Bacterial alkaline phosphatase and T4 DNA ligase were purchased from Bethesda Research Laboratories, T4 polynucleotide kinase was from P-L Biochemicals, and DNA polymerase I (Kornberg enzyme and the large fragment according to Klenow) was from Boehringer Mannheim. [y-32P]ATP (3000 Ci/ mmol; 1 Ci = 3.7 X 10'°becquerels), [a-32P]

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confidence: 99%

Expression of herpes simplex virus type I thymidine kinase gene in Escherichia coli.

Garapin¹,

Colbère-Garapin²,

Cohen-Solal³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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“…Herpesvirus TKs differ from cell cytosolic TKs in both their substrate specificity and the phosphate donors that they can utilize. Herpesvirus and certain mitochondrial TKs have the ability to use CTP as an alternative phosphate donor, but differ in their sensitivity to dCTP inhibition and physical properties (Kit, 1985;Kit et al, 1974).…”

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Evolution of herpesvirus thymidine kinases from cellular deoxycytidine kinase

Harrison

Thompson

Davison

1991

Journal of General Virology

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The thymidine kinases encoded by herpesviruses of higher vertebrates form a distinct group and are unrelated to the thymidine kinases (TKs) of other organisms. Their evolutionary source has not been identified, but our analysis has revealed a clear relationship with a sequence of human deoxycytidine kinase (dCK) published recently. We report the sequence of the putative TK of channel catfish virus, a herpesvirus of a lower vertebrate, and show that it is also related to dCK. We propose, therefore, that the TKs of herpesviruses of higher and lower vertebrates have evolved, either independently or successively, from a cellular dCK.

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“…After 3-hr incubation at 37°C, samples were spotted on Whatman DE-81 paper disks, which were washed four times in ethanol and assayed for radioactivity. For neutralization, 3 Ml of control serum or purified IgG against HSVl-infected rabbit cells that specifically neutralized HSV TK [a generous gift of D. R. Dubbs (17)] were added to 15 Ml of each enzyme sample, which contained 2-6 mg of protein per ml (18). The samples were incubated overnight at 4°C, clarified at 12,000 X g for 10 min, and assayed for TK activity.…”

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confidence: 99%