Expression ofHerpesSimplex VirusType1DNA Polymerase in Saccharomyces cerevisiae andDetection ofVirus-Specific EnzymeActivity inCell-Free Lysates

Theherpes simplex virus type1 (HSV-1) (strain 17)DNA polymerase gene hasbeenclonedintoan Escherichia coli-yeast shuttle vectorfused tothegalactokinase gene (GAL-I) promoter.Genescontrolled bythe GAL-Ipromoter areinduced bygalactose, uninduced byraffinose, andrepressed byglucose. Cell extracts from astrain ofSaccharomyces cerevisiae harboring this vector(Y-MH202, expressercells) grown inthepresence of galactose andassayed inhighsalt (100mM ammoniumsulfate) contained a novelDNA polymerase activity. No significant high-salt DNA polymerase activity was detected inextracts fromexpressercells grown inthe presenceofraffinose or inextracts fromcontrol cells containing theE.coli-yeast shuttle vectorwithout the HSV-1DNA polymerase gene grown inthepresenceofraffinose or galactose. Immunoblot analysis ofthecell extracts byusing a polyclonal rabbit antiserum prepared against a highly purified HSV-1DNA polymerase preparation revealed thespecific induction oftheHSV-1-140-kilodalton DNA polymerase polypeptide in expressercells grown ingalactose. Extracts fromthesame cells grown inraffinose or control cells grown in either raffinose orgalactose didnotcontain this immunoreactive polypeptide. Thehigh-salt DNA polymerase activity intheextracts fromexpressercells grown ingalactose was inhibited >90% byeither acyclovir triphosphate or aphidicolin, asexpected forHSV-1DNA polymerase. Inaddition, thehigh-salt polymerase enzyme activity could bedepleted fromextracts byimmunoprecipitation byusing purified immunoglobulin G fromthis same polyclonal rabbit antiserum. Theseresults demonstrate thesuccessful expression offunctional