Identification and structural analysis of the thymidine kinase gene of infectious bovine rhinotracheitis virus

In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase ge (tk), a well regulated viral gene, has been cloned in Escherichia coli HB101 by integrating partially Sau 3A-digested DNA fragments into a cosmid vector, pJB8. Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization. Results showed that this plasmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then transformed to E. coli tdk- mutant strains, Ky895 or C600 tdk- for the selection of the IBRV tk gene. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and sequences complementing the tk activity were isolated by subcloning. The E. coli mutant strain mutant strain C600 tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of [3H]thymidine into bacterial DNA over that of the C600 tdk- mutant. The plasmid, pIBRTK was also used as a probe to examine the expression of IBRV-tk gene in infected MDBK cells. A species of mRNA from infected cells with molecular weight of 2.2 kb was obtained when the total RNA or polyA-enriched RNA were electrophoresized in agarose gels and hybridized with 32P-pIBRTK DNA