Location, Cloning and Restriction Mapping the Thymidine Kinase Gene from Rice Strain Pseudorabies Virus
Koshy, Thomas I.
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The pseudorabies virus (PRV) i a porcine herpesvirus that lethally infects most mammals excluding the higher primates. While the virus may not be lethal to swine, it generates millions of dollars in economic losses annually. for use as a genetic marker it is of interest to clone the PRV thymidine kinase (TK) gene from the virus genome. The gene codes for the enzyme thymidine kinase which is responsible for the salvage phosphorylation of thymidine. This phosphorylation is an easily assayed quality and makes the TK gene very attractive as a genetic marker. Once cloned, the gene can be inserted into other PRV genes. In this way the importance of a given gene to successful PRV infection can be determined by disrupting the gene of interest with the TK gene, checking for TK activity to ensure TK insertion, and monitoring PRV infection. Most of the PRV, Rice Strain, genome has been examined without successfully locating the TK gene (L.E. Post, personal communication). Two fragments of the Rice strain PRV genome were cloned into the plasmid pBR322 and named pTK8 and pTK11. These were assayed for their ability to recombine with (TK-) PRV and recover (TK+)PRV. One of these plasmids, pTK11, did recover TK activity. A restriction map of the plasmid was then generated and the fragment in pTK11 subcloned.