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/sup -/ mutant strains, Ky895 or C600 tdk/sup -/ 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/sup -/ harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of (/sup 3/H)thymidine into bacterial DNA over that of the C600 tdk/sup -/ 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 /sup 32/P-pIBRTK DNA.
- Research Organization:
- California Univ., Davis (USA)
- OSTI ID:
- 6586459
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AZINES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CATTLE
CLONING
DAYS LIVING RADIOISOTOPES
DISEASES
DNA SEQUENCING
DNA-CLONING
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
GENES
HETEROCYCLIC COMPOUNDS
INFECTIOUS DISEASES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
MUTANTS
NUCLEI
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PATHOGENESIS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PYRIMIDINES
RADIOISOTOPES
RIBOSIDES
RNA
RUMINANTS
STRUCTURAL CHEMICAL ANALYSIS
THYMIDINE
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VERTEBRATES
VIRAL DISEASES