Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA

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Abstract
A selective plaque assay that uses thymidine kinase (TK)-deficient human 143 cells was developed to titer mixtures of TK+ and TK- vaccinia virus. This assay showed that methotrexate-resistant TK+ virus was formed in cells coinfected with TK- virus and wild-type virus DNA. By substituting vaccinia DNA fragments cloned in plasmids for virion DNA, this marker rescue system provided the basis for mapping the TK gene. Of the 15 HindIII fragments, only J could rescue 5 independently derived TK- mutants. This 5000-base-pair (bp) fragment maps .apprx. 80,000 bp from the left-end of the 180,000-bp vaccinia genome. Marker rescue could be detected with .ltoreq. 18 ng of plasmid and was proportionate to DNA concentration. The resistance to methotrexate of the TK+ recombinants was due to TK synthesis. Evidence that the HindIII J fragment contains the structural TK gene and not a regulatory element was shown by the synthesis of active TK in a cell-free system programmed with mRNA selected by hybridization to the plasmid. Previous studies indicated that mRNA coding for 3 immediate early polypeptides with MW of 41,000, 21,000 and 17,000 map within HindIII J. The mapping of the easily selectable vaccinia virus TK gene now opens the way to genetic manipulations that should increase our understanding of vaccinia virus gene expression and facilitate the use of vaccinia virus as an efficient cloning vector for foreign genes.