Identification of the thymidine kinase gene of infectious bovine rhinotracheitis virus and its function in escherichia coli hosts

Abstract

In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase gene ( tk),a well regulated viral gene has been chosen for this study. A cosmid library of IBRV has been constructed in Escherichia coli HB101 by cloning partially Sau3A-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 cosmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then used to transform E. coli tdk − mutant strains, Ky895 or C600 tdk − for the selection of the IBRV tk gene. The clones able to grow on the selection plates containing 5-fluorouracil, uridine, thymidine and ampicillin were selected and further characterized. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and the sequences complementing the tk activity have been isolated by subcloning. The plasmid, pIBRTK, was shown to grow on selection plates and therefore, retained the ability to complement the tk gene. The E. coli 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 C600 tdk − mutant.