Thymidine Kinases Induced by Avian and Human Herpesviruses

Kit, Saul; Jorgensen, George N.; Leung, Wai-Choi; Trkula, David; Dubbs, D. R.

doi:10.1159/000149438

Cited by 34 publications

(28 citation statements)

References 15 publications

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“…Formation of HSV TK activity has previously been studied in cells productively infected or biochemically transformed by HSV [1,2]. Detection of labeled HSV TK polypeptides has also been described after SDS-PAGE of total lysates of HSV-infecied cells [3], Because of the large number of labeled polypeptides present in crude lysates in the latter procedure, the labeling of HSV TK polypeptides might be obscured by radio-…”

Section: Discussionmentioning

confidence: 99%

Detection of Herpes Simplex Virus Thymidine Kinase Polypeptides in Cells Labeled with <sup>35</sup>S-Methionine

Kit¹,

Jorgensen²,

Dubbs³

et al. 1978

Intervirology

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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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Section: Discussionmentioning

confidence: 99%

Detection of Herpes Simplex Virus Thymidine Kinase Polypeptides in Cells Labeled with <sup>35</sup>S-Methionine

Kit¹,

Jorgensen²,

Dubbs³

et al. 1978

Intervirology

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“…Isozyme analyses by electrophoretic methods Fig. 1 shows representative examples of thymidine kinase analyses after disc electrophoresis in polyacrylamide gel (17,18). There is some overlap in cytosol thymidine kinase activity from human and mouse origin.…”

Section: Resultsmentioning

confidence: 99%

Cotransfer of two linked human genes into cultured mouse cells.

Willecke¹,

Lange²,

Kruger³

et al. 1976

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

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“…After one cycle of centrifugation in NaI gradients the percentage of viral DNA was increased to about 80%, and after the second cycle it was more than 95%. After removal of the ethidium bromide (11), the DNA was dialyzed extensively against 10 Davis (16) except that, following Kit et al (17), the gels and cathode buffer contained 0.2 mM thymidine and 2 mM ATP. After electrophoresis for 100 min at 3 mA per gel and 40, the gels were fractionated into 2-mm-thick slices that were assayed for TK activity as above (15) treated with DNA in the absence of Ca2+ or with DNA predigested with DNase, ng surviving colonies were observed.…”

Section: Methodsmentioning

confidence: 99%

Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA.

Bacchetti¹,

Graham²

1977

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

225

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Transformation of human cells from a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75)negative to a thymidine kinase-positive phenotype has been achieved by using pd DNA from herpes simplex virus type 2. The specific activityo tLieDNA was in the range 0.5 to 2.0 transformants per jig and the efficiency of gene transfer was up to 1 transformant per 105 recipient cells. Several transformed lines able to grow continuously in medium selective for thymidine kinase-positive cells have been established. All of these lines express a thymidine kinase activity of viral origin but they differ from each other in the stability of enzyme expression. Subclones derived from a given transformed line inherited the degree of stability of the parental line. The ability to use pure DNA to transfer genes between cultured mammalian cells would be of considerable importance to studies on the genetics of higher organisms. Although the transfer of viral genes that induce oncogenic transformation of cultured cells can be achieved routinely with purified viral DNA (1) and is likely to involve events similar to those expected to occur in the transfer of other types of genes, neither the viral gene products responsible for oncogenic transformation nor their functions are well defined. Consequently, oncogenic transformation is not a suitable model system for studying the characteristics of gene transfer in general. However, it is well established that herpes simplex viruses (HSV) code for a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) (TK) (2) and that this enzyme can be transferred to thymidine kinase-deficient (TK-) cells by transformation with UV-inactivated HSV-1 or HSV-2 (3) or with temperaturesensitive mutants of these viruses (4). The viral TK is well characterized and can be easily distinguished from most mammalian cell TKs (2). Moreover, a number of TK-mammalian cell lines are available as recipients for the viral enzyme. Thus, the transfer of TK would appear to be an excellent model system for studying gene transfer between mammalian cells.Our aim is to learn how viral genes are processed by recipient mammalian cells-i.e., whether and how they become integrated into the recipient cell genome, how their expression is controlled, and what determines the efficiency of these processes. Studies such as these hopefully may lead to DNA-mediated transfer of genes between mammalian cells as well as to a better understanding of some of the processes involved in oncogenic transformation. In this article we describe the transfer of the HSV-2 TK to human TK-cells by infection with purified HSV-2 DNA and the preliminary characterization of the resulting transformed lines. Asg/ml (a-BrdUrd)-selective for TK-cells; in this case, the lines were first either extensively washed free of HAT medium or were cultured for at least one passage in a-MEM. All lines were routinely subcultured twice weekly with a split ratio of 1:6 to 1:8. Periodic checks of cultures for mycoplasma contamination (8) were negative...

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Thymidine Kinases Induced by Avian and Human Herpesviruses

Cited by 34 publications

References 15 publications

Detection of Herpes Simplex Virus Thymidine Kinase Polypeptides in Cells Labeled with <sup>35</sup>S-Methionine

Detection of Herpes Simplex Virus Thymidine Kinase Polypeptides in Cells Labeled with <sup>35</sup>S-Methionine

Cotransfer of two linked human genes into cultured mouse cells.

Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA.

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