Purification of vaccinia virus-induced thymidine kinase activity from [35S]methionine-labeled cells

Kit, Saul; Jorgensen, George N.; Liav, Avraham; Zaslavsky, V.

doi:10.1016/0042-6822(77)90490-1

Cited by 22 publications

(19 citation statements)

References 19 publications

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“…Previous experiments have shown that the HSV-1 TK synthesized in productively infected cells has a molecular weight of about 80,000 daltons and consists of two subunits of about 40,000 daltons (2,8,26). To investigate the size of the TK polypeptides synthesized in cells biochemically transformed by fragments of plasmid DNAs, LM(TK )/TF pMHl ES1 and LM(TK )/TF pMHl Hc2 cells were labeled with 35S-methionine and the TK activities were purified by (NH4)2S04 precipitation, preparative disc PAGE, a second (NH4)2S04 precipitation step, and by glycerol gradient centrifugation (26,27). …”

Section: Resultsmentioning

confidence: 99%

“…These observations together with the findings that LM(TK ) cells can be biochemically transformed by Hinc It-and Bgl II-cleaved plasmid DNAs and by a purified 1.2kbp EcoR I-Sma I fragment, which has barely enough genetic information to code for the 40,000 dalton HSV-1 TK polypeptide, raised the possibility that fused polypeptides, which are translated from viral and flanking cellular DNA sequences, or truncated polypeptides, could have been made in some biochemically transformed cells (29). (17,26,27). Extracts were treated at 40C with 0.06ml of 10% polymin P per ml of extract and the precipitated nucleic acids were removed by centrifugation.…”

Section: Discussionmentioning

confidence: 99%

“…Ammonium sulfate (243mg/ml) was added to each supernatant fraction, the suspension was stirred for 30 min at 4°C, the precipitate was collected by centrifugation, and redissolved in modified enzyme buffer [0.15M KC1, 0.003M 2-mercaptoethanol (ME), 0.01M Tris-HCl, pH 8, 2.5mM ATP, 1.25mM Mg2+, 0.2mM thymidine, 0.05M epsilon aminocaproic acid (EACA), and 5% glycerol]. The HSV-1 TK activity was then purified by preparative polyacrylamide gel electrophoresis at 4°C, as described previously (26,27), except that a 1.9% stacking gel was used together with the 5% separating gel and fractions were collected in tubes containing 0.5mg normal rabbit IgG. Fractions containing the HSV-1 TK activity were pooled, the HSV-1 TK activity was reprecipitated with ammonium sulfate, redissolved in modified enzyme buffer, and further purified by centrifugation in 10-30%(v/v) glycerol gradients (26).…”

Section: Discussionmentioning

confidence: 99%

“…After the protein A-Sepharose complexes had been washed six times with 0.5M LiCl, 0.1% ME, O.LM Tris, pH 9.0, and once with modified enzyme buffer, they were resuspended in 75p4 of gel electrophoresis sample buffer (62.5mM Tris, pH 7.0, 3% SDS, 5% ME, and 10% glycerol) and placed in boiling water for 3 min. Supernatant solutions were then analyzed by electrophoresis in SDS-polyacrylamide (10%) vertical slab gels and autoradiography (26,27). Molecular weights of radioactive polypeptide bands were estimated by comparing their electrophoretic mobilities with those of actin (45K) and the protein standards previously described (27).…”

Section: Discussionmentioning

confidence: 99%

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Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids

et al. 1980

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The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMHl, pMHlA, and pMH4. These plasmids contain a 1,920bp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMHl, pMHlA, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bgl II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TKe phenotype. A 1,230bp EcoR ISma I fragment purified from pMHl DNA (and from plasmid pAGO DNA, the parent of pMH1) also transformed LM(TKI) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells was HSV-l-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.lkbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMHl DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids

et al. 1980

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“…The resistance of HCMV to acyclovir appears to be due primarily to the absence of a virus-specified thymidine kinase (9). Vaccinia virus, on the other hand, induces a thymidine kinase (15,16). However, Fyfe et al (12) showed that the thymidine kinase induced by vaccinia virus was unable to phosphorylate acyclovir.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of cellular alpha and virally induced deoxyribonucleic acid polymerases by the triphosphate of acyclovir

Clair¹,

Furman²,

Lubbers³

et al. 1980

Antimicrob Agents Chemother

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The effect of the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine) on cellular a deoxyribonucleic acid (DNA) polymerases (DNA nucleotidyltransferases), DNA polymerases of several members of the herpes group, vaccinia virus DNA polymerase, and Friend leukemia virus ribonucleic acid-dependent DNA polymerase was examined. Several viruses, which were found to be susceptible to acyclovir, were found to induce DNA polymerases which were sensitive to acyclovir triphosphate (acyclo-GTP). Human cytomegalovirus and the H29R strain of herpes simplex virus type 1, however, were found to be relatively insusceptible to acyclovir, even though their induced DNA polymerases were inhibited by low concentrations of acyclo-GTP. The amount of acyclovir anabolized to acyclo-GTP was significantly lower for human cytomegalovirus and H29R than for the more susceptible viruses. Vaccinia virus and Friend leukemia virus induced DNA polymerases which were insensitive to inhibition by low concentrations of acyclo-GTP, anabolized little acyclovir to acyclo-GTP, and were found to be insensitive to inhibition by acyclovir. Uninfected WI-38 cells were not susceptible to inhibition by acyclovir, anabolized little acyclovir to acyclo-GTP, and had an a DNA polymerase which was insensitive to inhibition by low concentrations of acyclo-GTP.Considerable effort has been devoted to the discovery of chemical compounds which inhibit viral replication without affecting the normal cell functions. Elion et al. (8) and Schaeffer et al. (19) reported that the purine analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine) is a potent antiherpetic agent which possesses extremely low cytotoxicity to uninfected cells. Acyclovir is an inhibitor of herpes simplex virus (HSV) deoxyribonucleic acid (DNA) synthesis, whereas the synthesis of Vero cell DNA is only partially inhibited at acyclovir concentrations several-hundred-fold greater than the 50% effective dose (ED5o) for HSV type 1 (HSV-1) (11).Acyclovir is anabolized to the monophosphate form by the viral thymidine kinase (8, 12), after which it is converted to the diphosphate form by cellular guanosine monophosphate kinase (W. H. Miller and R. L. Miller, J. Biol. Chem., in press) and then to the triphosphate form, apparently by cellular enzymes. Elion et al. (8) and Furman et al. (11) reported that acyclovir triphosphate (acyclo-GTP) is an inhibitor of HSV-1 DNA polymerases (DNA nucleotidyltransferases) and, to a lesser extent, of cellular a DNA polymerases.This report examines the ability of acyclo-GTP to inhibit the activity of several virus-induced and two cellular a DNA polymerases. The viruses represented include HSV-1 and -2, human cytomegalovirus (HCMV), vaccinia virus, and an avian ribonucleic acid (RNA) tumor virus. Correlations are made between the degrees of sensitivity of the enzymes to acyclo-GTP, ED5o's (expressed as micromolar acyclovir) for the viruses and cells, and amounts of acyclovir anabolized to acyclo-GTP by the virus-infected and uninfected cells.

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Structure and function of vaccinia virus thymidine kinase: Biomedical relevance and implications for antiviral drug design

Black

Hruby

1991

Reviews in Medical Virology

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Purification of vaccinia virus-induced thymidine kinase activity from [35S]methionine-labeled cells

Cited by 22 publications

References 19 publications

Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids

Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids

Inhibition of cellular alpha and virally induced deoxyribonucleic acid polymerases by the triphosphate of acyclovir

Structure and function of vaccinia virus thymidine kinase: Biomedical relevance and implications for antiviral drug design

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