Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Oct;15(10):5322–5328. doi: 10.1128/mcb.15.10.5322

A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice.

B Salomon 1, S Maury 1, L Loubière 1, M Caruso 1, R Onclercq 1, D Klatzmann 1
PMCID: PMC230780  PMID: 7565681

Abstract

Dividing eukaryotic cells expressing the herpes simplex virus type 1 thymidine kinase (TK) gene are sensitive to the cytotoxic effect of nucleoside analogs such as acyclovir or ganciclovir (GCV). Transgenic mice with cell-targeted expression of this conditional toxin have been used to create animals with temporally controlled cell-specific ablation. In these animal models, which allow the study of the physiological importance of a cell type, males are sterile. In this study, we showed that this phenomenon is due to testis-specific high-level expression of short TK transcripts initiated mainly upstream of the second internal ATG of the TK gene. This expression is DNA methylation independent. To obtain a suicide gene that does not cause male infertility, we generated and analyzed the properties of a truncated TK (delta TK) lacking the sequences upstream of the second ATG. We showed that when expressed at sufficient levels, the functional properties of delta TK are similar to those of TK in terms of thymidine or GCV phosphorylation. This translated into a similar GCV-dependent toxicity for delta TK- or TK-expressing cells, both in vitro and in transgenic mice. However, delta TK behaved differently from TK in two ways. First, it did not cause sterility in delta TK transgenic males. Second, low-level delta TK RNA expression did not confer sensitivity to GCV. The uses of delta TK in cell-specific ablation in transgenic mice and in gene therapy are discussed.

Full Text

The Full Text of this article is available as a PDF (352.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Al-Shawi R., Burke J., Jones C. T., Simons J. P., Bishop J. O. A Mup promoter-thymidine kinase reporter gene shows relaxed tissue-specific expression and confers male sterility upon transgenic mice. Mol Cell Biol. 1988 Nov;8(11):4821–4828. doi: 10.1128/mcb.8.11.4821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ariel M., McCarrey J., Cedar H. Methylation patterns of testis-specific genes. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2317–2321. doi: 10.1073/pnas.88.6.2317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arya S. K., Guo C., Josephs S. F., Wong-Staal F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science. 1985 Jul 5;229(4708):69–73. doi: 10.1126/science.2990040. [DOI] [PubMed] [Google Scholar]
  4. Black M. E., Loeb L. A. Identification of important residues within the putative nucleoside binding site of HSV-1 thymidine kinase by random sequence selection: analysis of selected mutants in vitro. Biochemistry. 1993 Nov 2;32(43):11618–11626. doi: 10.1021/bi00094a019. [DOI] [PubMed] [Google Scholar]
  5. Borrelli E., Heyman R. A., Arias C., Sawchenko P. E., Evans R. M. Transgenic mice with inducible dwarfism. Nature. 1989 Jun 15;339(6225):538–541. doi: 10.1038/339538a0. [DOI] [PubMed] [Google Scholar]
  6. Boyes J., Bird A. Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein. EMBO J. 1992 Jan;11(1):327–333. doi: 10.1002/j.1460-2075.1992.tb05055.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brady H. J., Miles C. G., Pennington D. J., Dzierzak E. A. Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):365–369. doi: 10.1073/pnas.91.1.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Braun R. E., Lo D., Pinkert C. A., Widera G., Flavell R. A., Palmiter R. D., Brinster R. L. Infertility in male transgenic mice: disruption of sperm development by HSV-tk expression in postmeiotic germ cells. Biol Reprod. 1990 Oct;43(4):684–693. doi: 10.1095/biolreprod43.4.684. [DOI] [PubMed] [Google Scholar]
  9. Caruso M., Klatzmann D. Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):182–186. doi: 10.1073/pnas.89.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Caruso M., Panis Y., Gagandeep S., Houssin D., Salzmann J. L., Klatzmann D. Regression of established macroscopic liver metastases after in situ transduction of a suicide gene. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7024–7028. doi: 10.1073/pnas.90.15.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cedar H. DNA methylation and gene activity. Cell. 1988 Apr 8;53(1):3–4. doi: 10.1016/0092-8674(88)90479-5. [DOI] [PubMed] [Google Scholar]
  12. Coen D. M., Irmiere A. F., Jacobson J. G., Kerns K. M. Low levels of herpes simplex virus thymidine- thymidylate kinase are not limiting for sensitivity to certain antiviral drugs or for latency in a mouse model. Virology. 1989 Feb;168(2):221–231. doi: 10.1016/0042-6822(89)90261-4. [DOI] [PubMed] [Google Scholar]
  13. Culver K. W., Blaese R. M. Gene therapy for cancer. Trends Genet. 1994 May;10(5):174–178. doi: 10.1016/0168-9525(94)90095-7. [DOI] [PubMed] [Google Scholar]
  14. Culver K. W., Ram Z., Wallbridge S., Ishii H., Oldfield E. H., Blaese R. M. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science. 1992 Jun 12;256(5063):1550–1552. doi: 10.1126/science.1317968. [DOI] [PubMed] [Google Scholar]
  15. Danos O., Mulligan R. C. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6460–6464. doi: 10.1073/pnas.85.17.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dragic T., Charneau P., Clavel F., Alizon M. Complementation of murine cells for human immunodeficiency virus envelope/CD4-mediated fusion in human/murine heterokaryons. J Virol. 1992 Aug;66(8):4794–4802. doi: 10.1128/jvi.66.8.4794-4802.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dzierzak E., Daly B., Fraser P., Larsson L., Müller A. Thy-1 tk transgenic mice with a conditional lymphocyte deficiency. Int Immunol. 1993 Aug;5(8):975–984. doi: 10.1093/intimm/5.8.975. [DOI] [PubMed] [Google Scholar]
  18. Field A. K., Davies M. E., DeWitt C., Perry H. C., Liou R., Germershausen J., Karkas J. D., Ashton W. T., Johnston D. B., Tolman R. L. 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine: a selective inhibitor of herpes group virus replication. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4139–4143. doi: 10.1073/pnas.80.13.4139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Furman P. A., McGuirt P. V., Keller P. M., Fyfe J. A., Elion G. B. Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Virology. 1980 Apr 30;102(2):420–430. doi: 10.1016/0042-6822(80)90109-9. [DOI] [PubMed] [Google Scholar]
  20. Fyfe J. A., Keller P. M., Furman P. A., Miller R. L., Elion G. B. Thymidine kinase from herpes simplex virus phosphorylates the new antiviral compound, 9-(2-hydroxyethoxymethyl)guanine. J Biol Chem. 1978 Dec 25;253(24):8721–8727. [PubMed] [Google Scholar]
  21. Geng Y., Johnson L. F. Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter. Mol Cell Biol. 1993 Aug;13(8):4894–4903. doi: 10.1128/mcb.13.8.4894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Graessmann A., Sandberg G., Guhl E., Graessmann M. Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation. Mol Cell Biol. 1994 Mar;14(3):2004–2010. doi: 10.1128/mcb.14.3.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haarr L., Flatmark T. Evidence that deletion of coding sequences in the 5' end of the thymidine kinase gene of herpes simplex virus type 1 affects the stability of the gene products. J Gen Virol. 1987 Nov;68(Pt 11):2817–2829. doi: 10.1099/0022-1317-68-11-2817. [DOI] [PubMed] [Google Scholar]
  24. Halpern M. E., Smiley J. R. Effects of deletions on expression of the herpes simplex virus thymidine kinase gene from the intact viral genome: the amino terminus of the enzyme is dispensable for catalytic activity. J Virol. 1984 Jun;50(3):733–738. doi: 10.1128/jvi.50.3.733-738.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Heyman R. A., Borrelli E., Lesley J., Anderson D., Richman D. D., Baird S. M., Hyman R., Evans R. M. Thymidine kinase obliteration: creation of transgenic mice with controlled immune deficiency. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2698–2702. doi: 10.1073/pnas.86.8.2698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huber B. E., Richards C. A., Krenitsky T. A. Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma: an innovative approach for cancer therapy. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8039–8043. doi: 10.1073/pnas.88.18.8039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Irmiere A. F., Manos M. M., Jacobson J. G., Gibbs J. S., Coen D. M. Effect of an amber mutation in the herpes simplex virus thymidine kinase gene on polypeptide synthesis and stability. Virology. 1989 Feb;168(2):210–220. doi: 10.1016/0042-6822(89)90260-2. [DOI] [PubMed] [Google Scholar]
  28. Iwakura Y., Asano M., Nishimune Y., Kawade Y. Male sterility of transgenic mice carrying exogenous mouse interferon-beta gene under the control of the metallothionein enhancer-promoter. EMBO J. 1988 Dec 1;7(12):3757–3762. doi: 10.1002/j.1460-2075.1988.tb03259.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kamogawa Y., Minasi L. A., Carding S. R., Bottomly K., Flavell R. A. The relationship of IL-4- and IFN gamma-producing T cells studied by lineage ablation of IL-4-producing cells. Cell. 1993 Dec 3;75(5):985–995. doi: 10.1016/0092-8674(93)90542-x. [DOI] [PubMed] [Google Scholar]
  30. Kremmer T., Paulik E., Boldizsár M., Holényi I. Application of the fast protein liquid chromatographic system and MonoQ HR 5/5 anion exchanger to the separation of nucleotides. J Chromatogr. 1989 Aug 25;493(1):45–52. doi: 10.1016/s0378-4347(00)82707-3. [DOI] [PubMed] [Google Scholar]
  31. Liu Q. Y., Summers W. C. Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. Virology. 1988 Apr;163(2):638–642. doi: 10.1016/0042-6822(88)90308-x. [DOI] [PubMed] [Google Scholar]
  32. McKnight S. L. The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. Nucleic Acids Res. 1980 Dec 20;8(24):5949–5964. doi: 10.1093/nar/8.24.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Minasi L. E., Kamogawa Y., Carding S., Bottomly K., Flavell R. A. The selective ablation of interleukin 2-producing cells isolated from transgenic mice. J Exp Med. 1993 May 1;177(5):1451–1459. doi: 10.1084/jem.177.5.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Monk M., Boubelik M., Lehnert S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development. 1987 Mar;99(3):371–382. doi: 10.1242/dev.99.3.371. [DOI] [PubMed] [Google Scholar]
  35. Moolten F. L. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res. 1986 Oct;46(10):5276–5281. [PubMed] [Google Scholar]
  36. Neznanov N., Thorey I. S., Ceceña G., Oshima R. G. Transcriptional insulation of the human keratin 18 gene in transgenic mice. Mol Cell Biol. 1993 Apr;13(4):2214–2223. doi: 10.1128/mcb.13.4.2214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roberts J. M., Axel R. Gene amplification and gene correction in somatic cells. Cell. 1982 May;29(1):109–119. doi: 10.1016/0092-8674(82)90095-2. [DOI] [PubMed] [Google Scholar]
  38. Salehi-Ashtiani K., Widrow R. J., Markert C. L., Goldberg E. Testis-specific expression of a metallothionein I-driven transgene correlates with undermethylation of the locus in testicular DNA. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8886–8890. doi: 10.1073/pnas.90.19.8886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Salomon B., Lorès P., Pioche C., Racz P., Jami J., Klatzmann D. Conditional ablation of dendritic cells in transgenic mice. J Immunol. 1994 Jan 15;152(2):537–548. [PubMed] [Google Scholar]
  40. Smiley J. R., Swan H., Pater M. M., Pater A., Halpern M. E. Positive control of the herpes simplex virus thymidine kinase gene requires upstream DNA sequences. J Virol. 1983 Aug;47(2):301–310. doi: 10.1128/jvi.47.2.301-310.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. St Clair M. H., Lambe C. U., Furman P. A. Inhibition by ganciclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information. Antimicrob Agents Chemother. 1987 Jun;31(6):844–849. doi: 10.1128/aac.31.6.844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sugden B., Marsh K., Yates J. A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus. Mol Cell Biol. 1985 Feb;5(2):410–413. doi: 10.1128/mcb.5.2.410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]
  44. Zemskova M. Y., Fodor I. Transient expression of deletion mutants of the herpes simplex virus thymidine kinase-encoding gene in mouse fibroblast cells. Gene. 1991 Oct 15;106(2):249–253. doi: 10.1016/0378-1119(91)90206-q. [DOI] [PubMed] [Google Scholar]
  45. al-Shawi R., Burke J., Wallace H., Jones C., Harrison S., Buxton D., Maley S., Chandley A., Bishop J. O. The herpes simplex virus type 1 thymidine kinase is expressed in the testes of transgenic mice under the control of a cryptic promoter. Mol Cell Biol. 1991 Aug;11(8):4207–4216. doi: 10.1128/mcb.11.8.4207. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES