Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates.
The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.
Haffey
M L
ML
Department of Microbial Biochemistry and Genetics, Squibb Institute for Medical Research, Princeton, New Jersey 08540-0130.
Stevens
J T
JT
Terry
B J
BJ
Dorsky
D I
DI
Crumpacker
C S
CS
Wietstock
S M
SM
Ruyechan
W T
WT
Field
A K
AK
D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000235:genetics*; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics
D004259:DNA-Directed DNA Polymerase; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI