dT Kinase Research Articles

Azidodeoxythymidine and didehydrodeoxythymidine as inhibitors and substrates of the human herpesvirus 8 thymidine kinase.

Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), encodes many core genes that have been maintained during evolution of the Herpesviridae. Among these is a thymidine kinase (TK) homologue (ORF21), which has 12% homology to the related TK encoded by herpes simplex virus. We show that the HHV-8 TK is a functional deoxythymidine (dT) kinase, with Michaelis constants (K(m)) for dT and ATP of 18.5 and 6.6 microM, respectively. Using homology modelling coupled with site-directed mutagenesis, we identify Gly265, Asp362 and Phe372 as key amino acid residues involved in the catalytic process. The HHV-8 TK is competitively inhibited by azidodeoxythymidine (zidovudine) and didehydrodeoxythymidine (stavudine) and can also accept these anti-retroviral compounds as substrates. These data have implications for our understanding of changes in AIDS-KS incidence following the clinical licensing of these compounds and in the development of new therapies for KS.

ChemInform Abstract: Zwitterionic 3′‐O‐Acyl Derivatives of Thymidine 5′‐Phosphate as Potential Sources of Intracellular Thymidine 5′‐Phosphate in Cells in Culture.

AbstractAusgehend von 5′‐O‐ Tritylthymidin (I) werden über die 3′‐Acylderivate (III) die Ammoniumchloride (V) und (VI) hergestellt, die in die zwitterionischen 5′‐Phosphate (VII) und (VIII) übergeführt werden.

Zwitterionic 3'-O-acyl derivatives of thymidine 5'-phosphate as potential sources of intracellular thymidine 5'-phosphate in cells in culture.

A convenient route is described for attachment of acyl groups CO(CH2)nN(Et)2(CH2)mNH(Et)2 (n = 3, m = 2; n = 4, m = 2-4), CO(CH2)nN(Et)2(CH2)mNEt3 (n = 4, m = 2-4), or CO(CH2)4N(CH2CH2)3N(CH2)nCH3 (n = 1 or 9) to O-3' of thymidine 5'-phosphate (TMP). The compounds are prototypes of 5'-nucleotide derivatives in which the two anionic charges could become partially masked in intramolecular anionic-cationic interactions and which might be able to diffuse into mammalian cells to furnish intracellular antimetabolite 5'-nucleotides by hydrolytic loss of a dicationic 3'-O-acyl group. At pH 7.6, 37 degrees C, hydrolyses of the 3'-ester linkages were pseudo first order with t1/2 values in the range 28-85 h. Paper chromatography in n-PrOH-H2O at pH 7.6 showed that type 1 or 2 derivatives were equally or slightly less hydrophobic than TMP (Rf 0.24), whereas the n-decyl type 3 compound (Rf 0.66) was markedly more hydrophobic, apparently because chain branching in group 3 is less than in 1 or 2. A sensitive and specific assay was developed for liberation of intracellular TMP in cultured mouse L fibroblasts in which synthesis of TMP and, hence, of DNA was suppressed by a combination of aminopterin and 5'-amino-5'-deoxythymidine (5'-NH2-dT). The TMP derivatives (100 microM) stimulated DNA synthesis, but omission of 5'-NH2-dT increased stimulation 6.5-fold, suggesting that stimulation occurred via degradation of the derivatives to dT. In confirmation, derivatives of type 2 (n = 4, m = 2 or 4) (100 microM) or type 3 (n = 9) (200 microM), in the presence of aminopterin, did not stimulate DNA synthesis in the LM(TK-) strain of L cells, which is genetically deficient in dT kinase. TMP (1 mM) stimulated DNA synthesis 2-3-fold and appeared to enter LM(TK-) cells without dephosphorylation, because dT (1 mM) gave no stimulation. If TMP is assumed to enter solely by passive diffusion, the inactivity of the TMP derivatives can be ascribed in part to their 2-fold higher molecular weight which can be expected to reduce flux through natural membranes ca. 16-fold.

Thymidine phosphorylating enzymes in Acetabularia: evidence for the occurrence of a thymidine kinase.

A test system allowing the estimation of the dT phosphorylating enzyme activity of one Acetabularia cell and of singular cell parts was developed. The occurrence in Acetabularia of a true dT kinase (EC. 2. 7. 1. 75) was demonstrated. This enzyme phosphorylates dT in the presence of ATP but not of AMP. dT kinase of Acetabularia is mainly bound to the particular fraction and a high percentage is localized outside the chloroplasts and mitochondria. After removal of the Triton X 100 (1%, by vol.) soluble part of the crude extract, a strong feedback inhibition by dTTP of the enzyme found in the pellet was observed. This enzyme may be solubilized with ionic detergents. The dT phosphorylating enzyme of the sediment (dT kinase) is distinct from supernatant phosphorylating enzyme in view of substrate specificity, stability, solubility, dTTP-inhibition, molecular mass, and K M value for dT.

Nucleo-cytoplasmic interactions in the regulation of thymidine phosphorylation in Acetabularia.

The activity of dT kinase (EC. 2. 7. 1. 75) was estimated during the development of nucleate and anucleate cells inAcetabularia. Enzyme activity increases as the cap attains its maximum diameter (Bannwarth & Schweiger 1975). Regulation of the dT kinase activity is observed even in the absence of the cell nucleus. Twelve to fifteen days after enucleation dT kinase activity increase sharply as the cap reaches its maximum diameter and decreases 5 days later. The increase is inhibited by puromycin, chloramphenicol, rifampicin and ethidium bromide but not by cycloheximide. These findings indicate that protein synthesis on 70S organelle ribosomes is involved in the regulation of the dT kinase. Since activities of mixtures from low activity cell extracts and from high activity cell extracts are additive, it is concluded that the regulation is not mediated through an activator protein but rather throughde novosynthesis of the enzyme protein.

A basis for bromodeoxyuridine resistance in plant cells

A mutant cell line (BU-54) of soybean ( Glycine max L. Merr) capable of growing at a high rate in liquid suspension culture in 1 mg/ml of 5-bromodeoxyuridine (BrdU) has been isolated. The BU-54 cells have an active uptake system for thymidine (dT) and BrdU, and possess dT kinase which utilizes both substrates. Cesium chloride analysis showed that the BU-54 cells incorporated very little BrdU into DNA unless 5-fluorodeoxyuridine (FdU) was present. The BU-54 showed a high degree of resistance to FdU and aminopterin. The cells also contained approximately three times more deoxythymidine monophosphate synthetase and 50 times more dihydrofolate reductase than sensitive wild type soybean cells. The results support the conclusion that the BrdU resistant line may overproduce deoxythymidine monophosphate which could result in a reduced incorporation of BrdU into cellular DNA.

Characterization of nucleoside phosphotransferase and thymidine kinase activities of chick embryo cells and of chick-mouse somatic cell hybrids

Experiments were carried out to characterize the thymidine (dT) phosphorylating activities of chick embryo, chick erythrocytes, and of chick mouse somatic cell hybrids derived from fused chick erythrocytes and dT kinase-deficient LM(TK) mouse cells. Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation analyses revealed that chick embryo cells contained four distinctive dT phosphorylating activities, two dT kinases and two nucleoside phosphotransferases. Thymidine kinase F. found principally in the cytosol, was also detected in mitochondrial and nuclear extracts, but was very low or absent from chick erythrocytes. Thymidine kinase A corresponds to the mitochondrial-specific isozyme found in bromodeoxyuridine-resistant mammalian cells. Nucleoside phosphotransferase activities were very active in chick embryo cytosol and were detected in embryo mitochondria! and nuclear extracts and cytosol and nuclear extracts of chick erythrocytes. Most of the chick embryo nucleoside phosphotransferase activity could be removed by purification of cytosol dT kinase F. Chick-mouse somatic cell hybrids exhibited chick dT kinase F, but neither chick dT kinase A. chick nucleoside phosphotransferase, nor mouse cytosol dT kinase activities. The results indicate (1) the genetic determinant for chick cytosol dT kinase F is on a different chromosome from the determinants for the chick nucleoside phosphotransferases and mitochondrial dT kinase A, and/or (2) only the chick cytosol dT kinase F, but neither the chick nucleoside phosphotransferases nor dT kinase A, was reactivated in the hybrids.

Analyses of deoxycytidylate deaminase molecular forms in human-mouse and monkey-mouse somatic cell hybrids.

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosal deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey and human and monkey enzymes can be separated. To learn whether the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. these findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybird cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable.

Distinctive properties of thymidine kinase isozymes induced by human and avian hepresviruses.

AbstractBiochemical and immunological experiments have been performed to learn whether human and avion herpesvirus‐induced dT kinases could be distinguished from the cytosol and mitochondrial dT kinase isozymes of uninfected cells. The dT kinases induced by human herpes simplex virus types 1 and 2 (HSV‐1 and HSV‐2) and by avian infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT) were used. The present experiments extend previous investigations and demonstrate that: (1) human and avian herpesvirus‐induced dT kinases differ from cytosol dT kinases of uninfected cells with respect to electrophoretic mobility, isoelectric point and phosphate donor specificity; (2) human and avian herpesvirus‐induced dT kinases differ from mitochondrial dT kinases of uninfected cells with respect to sedimentation coefficients and sensitivity to dCTP inhibition; and (3) HSV‐1‐ 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 dT kinase isozymes were all inhibited by the end product inhibitor, dTTP. Immunoglobulin (Ig) from sera of rabbits immunized with the HSV‐1‐induced dT kinase inhibited the homologous enzyme and some anti‐type 1 Ig preparations partially inhibited the HSV‐2 dT kinase, but the anti‐type 1 Ig inhibited neither cytosol and mitochondrial dT kinases from human, mouse, and monkey cells, nor the dT kinases induced by ILTV, HVT, or vaccinia virus. Anti‐type 2 Ig inhibited the homologous HSV‐2 dT kinase, but not HSV‐1 or human mitochondrial dT kinase.

GENETIC CONTROL OF MITOCHONDRIAL THYMIDINE KINASE IN HUMAN-MOUSE AND MONKEY-MOUSE SOMATIC CELL HYBRIDS

Distinctive thymidine (dT) kinase molecular forms are present in mouse, human, and monkey mitochondria. Disk polyacrylamide gel electrophoresis (disk PAGE) analyses have shown that the mitochondrial-specific dT kinases differ from cytosol dT kinases in relative electrophoretic mobilities (Rm). Furthermore, the mouse mitochondrial dT kinase differs in Rm value from primate mitochondrial dT kinases. The mouse and primate cytosol dT kinases can also be distinguished. Disk PAGE analyses have been carried out on the cytosol and mitochondrial dT kinases of human-mouse (WIL-8) and monkey-mouse (mK.CV(III)) somatic cell hybrids in order to learn whether the mitochondria of the hybrid cells contained murine mitochondrial-specific, primate mitochondrial-specific, or both dT kinases. WIL-8 cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse fibro blasts and from cytosol dT kinase-positive, mitochondrial dT kinase-positive human embryonic lung cells; they contained mostly mouse chromosomes and a few human chromosomes, including the determinant for human cytosol dT kinase. The mK.CV(III) cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse kidney cells and from cytosol dT kinase-positive, mitochondrial dT kinase-positive monkey kidney cells; they contained mostly mouse chromosomes and a few monkey chromosomes, including the determinant for monkey cytosol dT kinase. Disk PAGE analyses demonstrated that the mitochondria of human-mouse and monkey-mouse somatic cell hybrids contained the mouse-specific mitochondrial dT kinase but not the human- or monkey-specific mitochondrial dT kinase. These findings suggest that primate cytosol and mitochondrial thymidine kinase genes are coded on different chromosomes.

Gel electrophoresis and isoelectric focusing of mitochondrial and viral‐induced thymidine kinases

AbstractSubcellular fractionation studies have shown that the thymidine (dT) kinases induced by vaccinia and herpes simplex type 1 (HSV‐1) viruses are localized principally in the cytosol fraction. Isoelectric focusing, disc polyacrylamide gel electrophoresis (disc PAGE), and glycerol gradient centrifugation experiments were carried out in order to compare the viral‐induced enzymes with cellular dT kinases from the following sources: (1) cytosol fractions of dT kinase‐positive human (HeLa S3) and mouse (LM) cells; and (2) mitochondrial extracts of HeLa S3 and of bromodeoxyuridine‐resistant, dT kinase‐deficient HeLa (BU25) and LM (TK−) cells. These analyses revealed that the sedimentation coefficients of the vaccinia and HSV‐1 induced dT kinases were similar to those of the HeLa S3 and LM cytosol enzymes, but the viral‐induced dT kinases exhibited greater disc PAGE mobilities (Rm values) and lower pI values than the HeLa S3 and LM cytosol dT kinases. The vaccinia and HSV‐1 induced dT kinases were distinctly different. Both viral‐induced enzymes also differed from LM (TK−) mitochondrial dT kinase in sedimentation coefficient, Rm and pI. Small differences were found between the HSV‐1 induced dT kinase and a human mitochondrial‐specific isozyme. However, the HSV‐1 induced dT kinase resembled the mitochondrial‐specific human and mouse dT kinases in ability to substitute GTP, UTP and CTP for ATP as phosphate donors in the dT kinase reaction. In contrast, the LM and HeLa S3 cytosol dT kinases and the vaccinia‐induced dT kinase efficiently utilized only ATP as a phosphate donor. Recovery of LM and HeLa S3 cytosol dT kinase activities from gels was very poor when ribonucleoside 5'‐triphosphates were omitted from the upper buffer solution during disc PAGE. Also, in the absence of ATP, enzyme recoveries of mitochondrial and viral‐induced dT kinases were reduced and Rm values were decreased.

Distinctive molecular forms of thymidine kinase in mitochondria of normal and bromodeoxyuridine-resistant HeLa cells.

Disc polyacrylamide‐gel electrophoresis analyses have been carried out on the dT kinase activities of “cytosol” (high‐speed supernatant) and mitochondrial fractions of parental HeLa S3 and enzyme‐deficient HeLa(BU25) cells. The HeLa S3 cytosol fraction contained a very active dT kinase molecular form with a mobility relative to the tracking dye (Rx) of about 0.25. Conversion of HeLa S3 cells to dT‐kinase‐deficient HeLa(BU25) resulted in the loss of most of the cytosol dT kinase activity. The mitochondrial fraction of HeLa(BU25) cells did not contain the 0.25 Rx activity, but did shown 2 more rapidly migrating molecular forms (Rx of 0.4 and 0.6–0.7). The mitochondrial fraction of parental HeLa S3 cells also exhibited the 0.4 and 0.6 to 0.7 Rx dT kinase activities, and addition, contained the 0.25 Rx cytosol‐like enzyme. The distinctive dT kinase molecular forms observed in the mitochondrial fraction account for most of the residual enzyme activity of mutant HeLa(BU25) cells.

Properties of mitochondrial thymidine kinases of parental and enzyme-deficient HeLa cells

HeLa(BU25), a mutant subline of HeLa S3 cells, contains mitochondrial thymidine (dT) kinase, despite a marked deficiency in the dT kinase activity of the “cytosol” (high-speed supernatant) cell fraction. The HeLa(BU25) mitochondrial dT kinase differs from the “cytosol” enzyme of parental HeLa S3 cells in sedimentation coefficient, ability to utilize ribonucleoside 5′-triphosphates other than ATP as phosphate donors, sensitivity to inhibition by dCTP, and in disc polyacrylamide gel electrophoretic (disc PAGE) patterns. Two dT kinase activities [relative mobilities (Rm) of 0.4 and 0.6–0.7] were detected after disc PAGE of HeLa(BU25) mitochondrial extracts and both activities migrated more rapidly than the typical cytosol enzyme (Rm = 0.2) of dT kinase-positive human cells. The 0.6 to 0.7-Rm dT kinase of HeLa(BU25) mitochondria, but not the 0.4-Rm activity, utilized GTP and UTP, as well as ATP, as phosphate donors. HeLa S3 mitochondrial fractions contained the 0.6–0.7 Rm and the 0.4-Rm activities, and in addition, a “cytosol-like” 0.2-Rm activity. The 0.6 to 0.7-Rm dT kinase of HeLa S3 mitochondria utilized either UTP or ATP as phosphate donors, but the 0.4- and 0.2-Rm dT kinases utilized only ATP. Similarly, the HeLa S3 cytosol dT kinase efficiently utilized ATP, but not UTP, as a phosphate donor.

Distinctive properties of mitochondrial thymidine(dT)kinase from bromodeoxyuridine(dBU)-resistant mouse lines

dBU-resistant mouse lines lack detectable dT kinase activity in the high speed supernatant (cytosol) cell fraction. However, they contain a mitochondrial dT kinase, which sediments more slowly in glycerol gradients than the cytosol enzyme of parental mouse lines, exhibits a disc PAGE mobility relative to the tracking dye (Rm) of about 0.7–0.8, and utilizes ATP, UTP, GTP, and CTP as phosphate donors. The mitochondrial fraction of parental cells also contains this 0.7–0.8 Rm activity and, in addition, a minor dT kinase activity which migrates faster than the cytosol enzyme, but utilizes only ATP as phosphate donor. The cytosol dT kinase of parental mouse lines exhibits an Rm of about 0.2–0.3 and utilizes only ATP as phosphate donor.

Thymidine Kinases Induced by Avian and Human Herpesviruses

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.

Gel electrophoresis, isoelectric focusing, and localization of thymidine kinase in normal and simian virus 40-infected monkey cells.

Following SV40 infection of CV-1 monkey cells, cytosol thymidine (dT) kinase activity increased 4- to 10-fold. This enzyme was not significantly different from the cytosol dT kinase of uninfected cells with respect to mobility, isoelectric point, sedimentation coefficient, and phosphate donor specificity. Monkey mitochondria contain a distinctive dT kinase isozyme localized in the mitochondrial matrix. The distinctive mitochondrial isozyme had a higher electrophoretic mobility, lower isoelectric point, and smaller sedimentation coefficient than the cytosol dT kinase, and could utilize either ATP or UTP as phosphate donor; it did not increase after SV40 infection. Mitochondria also contained a cytosol-like dT kinase which was enhanced in infected cells. These findings suggest that SV40 infection derepresses the normal cytosol dT kinase of host cells.

Mitochondrial thymidine kinase of bromodeoxyuridine-resistant, kinase-deficient HeLa(BU25) cells

Total cell extracts of HeLa(BU25), a mutant subline of HeLa S3, are deficient in dT kinase activity. In contrast to the parental HeLa S3 cells, the nuclear DNA of HeLa(BU25) does not exhibit a “heavy” density after the HeLa(BU25) cells are grown in medium with dBU. Despite the loss of the principal dU-dT phosphorylating enzyme, HeLa(BU25) cells contain a mitochondrial dU-dT phosphorylating activity with a specific activity about equal to that of the HeLa S3 mitochondrial enzyme. Phosphorylation of 3H-dU by the mitochondrial enzyme requires ATP, is markedly inhibited by dTTP or nonradioactive dT, but not by Urd, Cyd, or Ado. Hence, it is a dT kinase. A nucleoside phosphotransferase, weakly active at pH 5.5–6.5, may also be present.

Effect of prolonged cultivation of SV40-transformed mouse cells in bromide-oxyuridine or pretreatment with mitomycin C on rescue of SV40.

AbstractA series of clonal lines (mKS‐U) of mouse kidney cells transformed by UV‐irradiated SV40 have been propagated serially in medium containing 5‐bromodeoxyuridine (dBU). Most of the mKS‐U lines became resistant to dBU during serial passage and became deficient in thymidine (dT) kinase activity. The dT kinase‐deficient mKS‐U lines were unable to grow in medium containing HATG (10−4 M hypoxanthine, 10−5 M aminopterin, 4 × 10−5 M thymidine, and 10−5 M glycine; Littlefield, 1965) because they were unable to utilize exogenous thymidine as a source of thymidylate when de novo synthesis of thymidylate was blocked by aminopterin. Growth in dBU medium did not increase the buoyant density of the DNA of mKS‐U lines deficient in dT kinase activity, demonstrating that dBU was not incorporated into the DNA of the cells. These observations show that a heritable change had occurred in the dBU‐grown mKS‐U cell lines. However, two dBU‐adapted cell lines, mKS‐U1 (BU) and mKS‐U2 (BU), did retain appreciable dT kinase activity even after 97 and 98 passages in dBU. The DNAs from mKS‐U1 (BU) and mKS‐U2 (BU) grown in dBU had buoyant densities greater than those of the DNAs from mouse cells grown in medium lacking dBU and the mKS‐U1 (BU) and mKS‐U2 (BU) lines were able to grow normally in HATG medium.Serial passage of poor, average, or good yielder transformed lines in dBU did not significantly change the frequency of induction of SV40 in fusion experiments with African green monkey kidney (CV‐1) cells. However, the SV40 rescued in fusion experiments with two poor yielders after serial passage in dBU tended to produce larger plaques than those rescued prior to passage in dBU. Serial passage in dBU did not permit rescue of SV40 from heterokaryons of the rare yielders or non‐yielders.The SV40 antigen was present in all the cell lines, even after prolonged cultivation in dBU, indicating that the SV40 genome was retained.Pretreatment of mKS‐U lines with mitomycin C prior to fusion with CV‐1 cells did not alter the virus yield from a good yielder, mKS‐U13, after fusion with CV‐1, and did not permit rescue of SV40 from rare or non‐yielder lines.

Virogenic properties of bromodeoxyuridine-sensitive and bromodeoxyuridine-resistant simian virus 40-transformed mouse kidney cells.

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.

Nonidentiy of some simian virus 40-induced enzymes with tumor antigen.

The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.