Electrophoresis of thymidine kinase activity synthesized by cells transformed by herpes simplex virus

Munyon, William; Buchsbaum, Robert; Paoletti, Enzo; Mann, Judith; Kraiselburd, Edmundo; Davis, Daniel B.

doi:10.1016/0042-6822(72)90525-9

Cited by 85 publications

(58 citation statements)

References 10 publications

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“…HSV DNA polymerase was assayed as described by Keir et al (1966) in the presence of 100 mM-ammonium sulphate to inhibit selectively cellular DNA polymerase. Thymidine kinase (TK) activity was assayed according to Munyon et al (1972). All enzymes were assayed under linear reaction conditions.…”

Section: Methodsmentioning

confidence: 99%

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Huszar

Beharry

Bacchetti

1983

Journal of General Virology

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SUMMARYWe have previously reported on the characterization of a novel ribonucleotide reductase induced in herpes simplex virus type 2 (HSV-2)-infected mammalian cells. The virus-induced enzyme was partially purified, free of the constitutive cell isozyme, by fractionation of infected cell extracts with ammonium sulphate. In this report we describe a further purification of the virus-induced enzyme and the development of a rabbit antiserum capable of specifically inhibiting its activity. Enzymically active salt fractions from infected cell extracts were sedimented through glycerol gradients; the virus-induced enzyme was found to sediment approximately 2.5 times faster than the constitutive, or control, enzyme and was separated from the majority of the protein in the sample. Immunization of rabbits with the partially purified enzyme preparation recovered from gradients resulted in the synthesis of antibodies which completely and specifically inhibited the virus-inducod reductase, and precipitated one major polypeptide and a few minor species from both HSV-1-and HSV-2-infected cells. The antibodies, however, exhibited much greater affinity for the HSV-2 than the HSV-1 antigen. These results demonstrate that the virus-induced enzyme differs antigenically, as well as biochemically, from the constitutive cell isozyme and lend further support to the hypothesis that the enzyme is virus-codod.

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Section: Methodsmentioning

confidence: 99%

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Huszar

Beharry

Bacchetti

1983

Journal of General Virology

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“…Viral TK also can be expressed in TK-cells which have been "biochemically transformed" to a TKV phenotype by infection with UV-inactivated virus (6,7) or viral DNA fragments (8). The TK gene is heritably and stably incorporated into the genome of the cell and is expressed in the apparent absence of the immediate-early viral gene products.…”

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confidence: 99%

Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1.

Wagner¹,

Sharp²,

Summers³

1981

Proc. Natl. Acad. Sci. U.S.A.

345

194

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“…The basis for the selection of transformed cells on HAT is that those cells in which the information for HSV-specific TK synthesis is stably incorporated will be able to utilize thymidine and multiply, whereas the XCTK-cells will not (13). Alternative pathways that would enable cells to bypass the aminopterin block are conceivable, so we tested the transformed lines for TK activity and determined the migration rate of TK activities by gel electrophoresis as described by Munyon et al (7). Total TK activity in extracts of HSV1 or HSV2-transformed cells was usually 6-15 times greater than that in extracts of XCTKcells.…”

mentioning

confidence: 99%

Transformation of Cultured Mammalian Cells by Viable Herpes Simplex Virus Subtypes 1 and 2

Garfinkle

McAuslan

1974

Proc. Natl. Acad. Sci. U.S.A.

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A continuous line of rodent cells carrying the genome of the Rous sarcoma virus was stably transformed by exposure to viable herpes simplex virus of either subtype 1 or 2. Transformation was accompanied by alteration of morphology, growth characteristics, acquisition of a thymidine kinase activity (EC 2.7.1.21) resembling the corresponding specific activity of the enzyme in the herpes simplex virus, and capacity to express continuously some antigens specific for herpes simplex virus.A considerable body of evidence implicates herpesvirus as the etiological agent of certain forms of cancer (1, 2). Of particular current interest is the possible link between human cervical carcinoma and herpes simplex virus (HSV) subtype 2, but the evidence to date is largely circumstantial. Cells transformed in culture by HSV might provide a valuable system to explore the oncogenic potential of this virus. Biochemical transformation of thymidine-kinase-deficient (TK-) mouse cells to the thymidine-kinase-positive phenotype has in fact been achieved using ultra-violet inactivated HSV (3). The molecular basis of the mode of selection of transformants and of the acquisition of new kinase function has yet to be rigorously established, although the available evidence is consistent with the conclusion that the cells stably inherit the capacity to synthesize thymidine kinase (TK) that is specific for HSV.To determine which biochemical properties of HSV most closely reflect its putative oncogenic function, it would be useful to have a cell system which could be transformed by viable virus or conditional lethal mutants thereof. We describe here the transformation of cells by HSV, subtype 1 Antiserum to rabbit-y-globulin was prepared by inoculating goats with purified rabbit-y-globulin (Miles Laboratories, St. Louis, Mo.). Goats were inoculated twice intradermally 220

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Electrophoresis of thymidine kinase activity synthesized by cells transformed by herpes simplex virus

Cited by 85 publications

References 10 publications

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Herpes Simplex Virus-induced Ribonucleotide Reductase: Development of Antibodies Specific for the Enzyme

Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1.

Transformation of Cultured Mammalian Cells by Viable Herpes Simplex Virus Subtypes 1 and 2

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