Mapping and identification of the vaccinia virus thymidine kinase gene

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Abstract
The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the rabbit reticulocyte cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains .apprx. 2.6% of the genome (4800 base pairs). This DNA fragment encodes 4 known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the MW range 15,000-25,000. Hybridization of these mRNA to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent MW of 19,000. The native MW of VV thymidine kinase is .apprx. 80,000 so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000 MW subunits.