Subcellular fractionation studies have shown that the thymidine (dT) kinases induced by vaccinia and herpes simplex type I ( H S V-I) viruses dT kinase-deficient HeLa (BU25) and L M ( TK-) cells. These analyses revealed that the sedimentation coefficients of the vaccinia and HSV-I induced dT kinases were similar to those of the HeLa S3 and L M cytosol enzymes, but the viralinduced dT kinases exhibited greater disc PAGE mobilities ( R m values) and lower p l values than the HeLa S3 and L M cytosol dT kinases. The vaccinia and HSV-I induced dT kinases were distinctly different. Both viral-induced enzymes also differed from L M ( T K -) mitochondrial dT kinase in sedimentation coefficient, R m and PI. Small differences were found between the H S V-I induced dT kinase and a human mitochondrialspecific isozyme. However, the HS V-1 induced dT kinase resembled the mitochondrialspecific human and mouse dT kinases in ability to

Biochemical and immui~olo~ical experiments have been performed to learn whether human and avian herpesvirus-induced d T kinases could be distinguished from the cytosol and mitochondrial d T kinase isozymes of uninfected cells. The dT kinases itiduced by human herpes simplex virus types I and 2 ( H S V-I and HSV-2) and by avian infectious laryngotracheitis virus ( I L T V ) and herpesvirus of turkeys ( H V T ) were kinases oj uninfected cells with respect to sedimentation coeficients and sensitivity to dCTP inhibition; and ( 3 ) HSV-I-and HSV-2-induced dT kinases differ from human cytosol and mitochondrial dT kinases in thermal lability and antigenic determinants. The cellular and the viral-induced d T kinase isozymes were all inhibited by the end product inhibitor, dTTP. Immunoglobulin ( l g ) from sera of rabbits immunized with the HS V-I-induced dTkinase inhibited the homologous enzyme and some anti-type 1 Ig preparations partial1.v inhibited the H S V-2 d T kinase, but the anti-type I Ig inhibited neither cytosol and mitochondrial d T kinases from human, mouse, and monkey cells, nor the d T kinases induced by ILTV, H V T , or vaccinia virus. Anti-type 2 Ig inhibited the homologous HSV-2 d T kinase, but not HSV-I or human mitochondria1 d T kinase.Thymidine (dT) kinase activity is induced early after infection of cells by avian, porcine, simian and human herpesviruses (Buchan and Watson, 1969;Kit et al., 1967 Kit et al., , 1974a Kit et al., , e, 1975 Thouless, 1972). The herpesviruses-induced enzymes differ from the major cytosol d T kinases of host cells in biochemical, immunological and genetic properties, suggesting that they are virus-coded proteins. Hence, the herpesvirus-induced d T kinases are potentially useful markers for: ( I ) studies on the regulation of gene expression; and (2) the detection of virus genetic information in transformed cells and in tumors suspected of having been caused by herpes-viruses. To be useful for these purposes, however, the herpesvirus-induced d T kinases must also be distinguishable from the genetically distinct mitochondrial d T kinase of host cells (Kit and Leung, 1974a, b ; Kit et al., 1972Kit et al., , 1973Kit et al., , 1974d. Previous studies have shown that the human and avian herpesvirus-induced d T kinases have larger sedimentation coefficients than vertebrate mitochondrial d T kinases. They also differ from mouse mitochondrial d T kinase in electrophoretic mobility and isoelectric point. However, the herpesvirus-induced d T kinases resemble chick, monkey and human mitochondrial d T kinases in phosphate donor specificity, electrophoretic mobility and isoelectric point. Therefore, they could easily be confused with mitochondrial d T kinases, particularly when the herpesvirus d T kinases are present at very low concentrations

Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation experiments were carried out to characterize thymidine (dT) kinase isozymes induced by herpesvirus of turkeys and infectious laryngotracheitis virus in the cytosol fraction of infected chick cells. The avian herpesvirus dT kinases differed from chick cytosol dT kinase in electrophoretic mobility, isoelectric point and phosphate donor specificity. The avian herpesvirus dT kinases resembled chick mitochondrial dT kinase in electrophoretic mobilityy and isoelectric point, but exhibited larger sedimentation coefficients. The avian herpesvirus enzymes closely resembled dT kinases induced by human herpes simplex viruses types 1 and 2.

To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5’-amino-5’-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.

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Cytosol thymidine kinase (TK) and deoxycytidylate (dCMP) deaminase formation was investigated in synchronized cultures of K12 Chinese hamster cells which have a temperature-sensitive lesion affecting the initiation of DNA synthesis. Enzyme formation was found to be cycloheximide-sensitive and also temperature-dependent. Beginning at about six hours after addition of medium with 10% calf serum to serum-depleted K12 cultures, cytosol TK and dCMP deaminase activities increased when the cultures were incubated at 36.5 degrees but not at 40.5 degrees. When cultures were shifted from 36.5 degrees to 40.5 degrees at 4,6, or 8 hours after serum addition, TK activity continued to increase, though not to the level observed at ten hours in cultures maintained at 36.5 degrees. Actinomycin D addition at the time of serum reversal or four hours later blocked the TK increase normally observed at the permissive temperature at ten hours. However, when actinomycin D addition was delayed for six or eight hours after serum addition, the increase in TK measured at ten hours resembled that observed in the temperature shift-up experiments. The results provide evidence that the mutation in K12 Chinese hamster cells most likely blocks the progression through G1 into S and suggest that transcription or post-transcriptional processing required for TK formation is affected.

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