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. 1988 Dec;8(12):5280–5291. doi: 10.1128/mcb.8.12.5280

Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells.

H B Lieberman 1, P F Lin 1, D B Yeh 1, F H Ruddle 1
PMCID: PMC365630  PMID: 3244356

Abstract

We previously isolated and characterized the structure of murine thymidine kinase (tk) genomic and cDNA sequences to begin a study designed to identify regions of the tk gene important for regulated expression during the transition of cells from G0 to a proliferating state. In this report, we describe the stable transfection of the cloned gene into L-M(TK-) cells and show that both thymidine kinase (TK) enzyme activity and DNA synthesis increase in parallel when transfectants in G0 arrest are stimulated by serum. To define promoter and regulatory regions more precisely, we have constructed a series of tk minigenes and have examined their expression in stable transfectants after serum stimulation. We have identified a 291-base-pair DNA fragment at the 5' end of the tk gene that has promoter function, and we have determined its sequence. In addition, we have found that DNA sequences which mediate serum-induced expression of TK are transcribed, since expression of the murine tk cDNA, fused to a promoter from either the murine tk gene, the simian virus 40 early region, or the herpes simplex virus tk gene, is stimulated by serum. Our constructs also reveal that the murine tk polyadenylation signal is not required for regulation, nor is most of the 3' untranslated region. RNA dot blot analysis indicates that murine cytoplasmic tk mRNA levels always parallel TK enzyme activity. Nuclear runon transcription assays show less than a 2-fold increase in transcription from the cloned tk gene in serum-stimulated transfectants, but an 11-fold increase in mouse L929 cells, which are inherently TK+. These results taken together suggest that the murine tk gene is controlled in serum-stimulated cells by a transcriptional mechanism influenced by DNA sequences that flank tk and also by a posttranscriptional system linked to gene sequences that are transcribed.

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Selected References

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  1. Artishevsky A., Wooden S., Sharma A., Resendez E., Jr, Lee A. S. Cell-cycle regulatory sequences in a hamster histone promoter and their interactions with cellular factors. 1987 Aug 27-Sep 2Nature. 328(6133):823–827. doi: 10.1038/328823a0. [DOI] [PubMed] [Google Scholar]
  2. Basilico C., Zouzias D. Regulation of viral transciption and tumor antigen expression in cells transformed by simian virus 40. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1931–1935. doi: 10.1073/pnas.73.6.1931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bello L. J. Regulation of thymidine kinase synthesis in human cells. Exp Cell Res. 1974 Dec;89(2):263–274. doi: 10.1016/0014-4827(74)90790-3. [DOI] [PubMed] [Google Scholar]
  4. Bradshaw H. D., Jr Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5588–5591. doi: 10.1073/pnas.80.18.5588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  6. Brown A. M., Jeltsch J. M., Roberts M., Chambon P. Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6344–6348. doi: 10.1073/pnas.81.20.6344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clayton D. A., Teplitz R. L. Intracellular mosaicism (nuclear - -mitochondrial + ) for thymidine kinase in mouse L cells. J Cell Sci. 1972 Mar;10(2):487–493. doi: 10.1242/jcs.10.2.487. [DOI] [PubMed] [Google Scholar]
  8. Colbere-Garapin F., Chousterman S., Horodniceanu F., Kourilsky P., Garapin A. C. Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3755–3759. doi: 10.1073/pnas.76.8.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  10. Coppock D. L., Pardee A. B. Control of thymidine kinase mRNA during the cell cycle. Mol Cell Biol. 1987 Aug;7(8):2925–2932. doi: 10.1128/mcb.7.8.2925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Denhardt D. T., Edwards D. R., Parfett C. L. Gene expression during the mammalian cell cycle. Biochim Biophys Acta. 1986 Oct 28;865(2):83–125. doi: 10.1016/0304-419x(86)90024-7. [DOI] [PubMed] [Google Scholar]
  12. Derman E., Krauter K., Walling L., Weinberger C., Ray M., Darnell J. E., Jr Transcriptional control in the production of liver-specific mRNAs. Cell. 1981 Mar;23(3):731–739. doi: 10.1016/0092-8674(81)90436-0. [DOI] [PubMed] [Google Scholar]
  13. Ellims P. H., Van der Weyden M. B., Medley G. Thymidine kinase isoenzymes in human malignant lymphoma. Cancer Res. 1981 Feb;41(2):691–695. [PubMed] [Google Scholar]
  14. Elsevier S. M., Kucherlapati R. S., Nichols E. A., Creagan R. P., Giles R. E., Ruddle F. H., Willecke K., McDougall J. K. Assignment of the gene for galactokinase to human chromosome 17 and its regional localisation to band q21-22. Nature. 1974 Oct 18;251(5476):633–636. doi: 10.1038/251633a0. [DOI] [PubMed] [Google Scholar]
  15. Gasser C. S., Schimke R. T. Cell cycle regulation of transfected murine dihydrofolate reductase genes. J Biol Chem. 1986 May 25;261(15):6938–6946. [PubMed] [Google Scholar]
  16. Gidoni D., Dynan W. S., Tjian R. Multiple specific contacts between a mammalian transcription factor and its cognate promoters. 1984 Nov 29-Dec 5Nature. 312(5993):409–413. doi: 10.1038/312409a0. [DOI] [PubMed] [Google Scholar]
  17. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  18. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  19. Groudine M., Casimir C. Post-transcriptional regulation of the chicken thymidine kinase gene. Nucleic Acids Res. 1984 Feb 10;12(3):1427–1446. doi: 10.1093/nar/12.3.1427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  21. Hofbauer R., Müllner E., Seiser C., Wintersberger E. Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA. Nucleic Acids Res. 1987 Jan 26;15(2):741–752. doi: 10.1093/nar/15.2.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. KIT S., DUBBS D. R., PIEKARSKI L. J., HSU T. C. DELETION OF THYMIDINE KINASE ACTIVITY FROM L CELLS RESISTANT TO BROMODEOXYURIDINE. Exp Cell Res. 1963 Aug;31:297–312. doi: 10.1016/0014-4827(63)90007-7. [DOI] [PubMed] [Google Scholar]
  23. Kasid A., Davidson N. E., Gelmann E. P., Lippman M. E. Transcriptional control of thymidine kinase gene expression by estrogen and antiestrogens in MCF-7 human breast cancer cells. J Biol Chem. 1986 Apr 25;261(12):5562–5567. [PubMed] [Google Scholar]
  24. Kaufman R. J., Sharp P. A. Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes. Mol Cell Biol. 1983 Sep;3(9):1598–1608. doi: 10.1128/mcb.3.9.1598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kellems R. E., Morhenn V. B., Pfendt E. A., Alt F. W., Schimke R. T. Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts. J Biol Chem. 1979 Jan 25;254(2):309–318. [PubMed] [Google Scholar]
  26. Kit S., Leung W. C., Jorgensen G. N., Trkula D., Dubbs D. R. Viral-induced thymidine kinase isozymes. Prog Med Virol. 1975;21:13–34. [PubMed] [Google Scholar]
  27. Kit S. Thymidine kinase, DNA synthesis and cancer. Mol Cell Biochem. 1976 Jun 15;11(3):161–182. doi: 10.1007/BF01744997. [DOI] [PubMed] [Google Scholar]
  28. Kozak C. A., Ruddle F. H. Assignment of the genes for thymidine kinase and galactokinase to Mus musculus chromosome 11 and the preferential segregation of this chromosome in Chinese hamster/mouse somatic cell hybrids. Somatic Cell Genet. 1977 Mar;3(2):121–133. doi: 10.1007/BF01551809. [DOI] [PubMed] [Google Scholar]
  29. Kreidberg J. A., Kelly T. J. Genetic analysis of the human thymidine kinase gene promoter. Mol Cell Biol. 1986 Aug;6(8):2903–2909. doi: 10.1128/mcb.6.8.2903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. LITTLEFIELD J. W. STUDIES ON THYMIDINE KINASE IN CULTURED MOUSE FIBROBLASTS. Biochim Biophys Acta. 1965 Jan 11;95:14–22. doi: 10.1016/0005-2787(65)90206-6. [DOI] [PubMed] [Google Scholar]
  31. Lewis J. A., Matkovich D. A. Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences. Mol Cell Biol. 1986 Jun;6(6):2262–2266. doi: 10.1128/mcb.6.6.2262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lewis J. A. Structure and expression of the Chinese hamster thymidine kinase gene. Mol Cell Biol. 1986 Jun;6(6):1998–2010. doi: 10.1128/mcb.6.6.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lin P. F., Lieberman H. B., Yeh D. B., Xu T., Zhao S. Y., Ruddle F. H. Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences. Mol Cell Biol. 1985 Nov;5(11):3149–3156. doi: 10.1128/mcb.5.11.3149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McBreen P., Orkwiszewski K. G., Chern C. J., Mellman W. J., Croce C. M. Synteny of the genes for thymidine kinase and galactokinase in the mouse and their assignment to mouse chromosome 11. Cytogenet Cell Genet. 1977;19(1):7–13. doi: 10.1159/000130789. [DOI] [PubMed] [Google Scholar]
  35. McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  36. McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
  37. Merrill G. F., Harland R. M., Groudine M., McKnight S. L. Genetic and physical analysis of the chicken tk gene. Mol Cell Biol. 1984 Sep;4(9):1769–1776. doi: 10.1128/mcb.4.9.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Merrill G. F., Hauschka S. D., McKnight S. L. tk Enzyme expression in differentiating muscle cells is regulated through an internal segment of the cellular tk gene. Mol Cell Biol. 1984 Sep;4(9):1777–1784. doi: 10.1128/mcb.4.9.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Miskimins W. K., Roberts M. P., McClelland A., Ruddle F. H. Protein interactions at the transferrin receptor gene promoter. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 2):725–729. doi: 10.1101/sqb.1986.051.01.085. [DOI] [PubMed] [Google Scholar]
  40. Morello D., Duprey P., Israel A., Babinet C. Asynchronous regulation of mouse H-2D and beta-2 microglobulin RNA transcripts. Immunogenetics. 1985;22(5):441–452. doi: 10.1007/BF00418090. [DOI] [PubMed] [Google Scholar]
  41. Navalgund L. G., Rossana C., Muench A. J., Johnson L. F. Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. J Biol Chem. 1980 Aug 10;255(15):7386–7390. [PubMed] [Google Scholar]
  42. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Page M. J., Parker M. G. Androgen-regulated expression of a cloned rat prostatic c3 gene transfected into mouse mammary tumor cells. Cell. 1983 Feb;32(2):495–502. doi: 10.1016/0092-8674(83)90469-5. [DOI] [PubMed] [Google Scholar]
  44. Pardee A. B., Dubrow R., Hamlin J. L., Kletzien R. F. Animal cell cycle. Annu Rev Biochem. 1978;47:715–750. doi: 10.1146/annurev.bi.47.070178.003435. [DOI] [PubMed] [Google Scholar]
  45. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  46. Roginski R. S., Skoultchi A. I., Henthorn P., Smithies O., Hsiung N., Kucherlapati R. Coordinate modulation of transfected HSV thymidine kinase and human globin genes. Cell. 1983 Nov;35(1):149–155. doi: 10.1016/0092-8674(83)90217-9. [DOI] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schlosser C. A., Steglich C., deWet J. R., Scheffler I. E. Cell cycle-dependent regulation of thymidine kinase activity introduced into mouse LMTK- cells by DNA and chromatin-mediated gene transfer. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1119–1123. doi: 10.1073/pnas.78.2.1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stewart C. J., Ito M., Conrad S. E. Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene. Mol Cell Biol. 1987 Mar;7(3):1156–1163. doi: 10.1128/mcb.7.3.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stuart P., Ito M., Stewart C., Conrad S. E. Induction of cellular thymidine kinase occurs at the mRNA level. Mol Cell Biol. 1985 Jun;5(6):1490–1497. doi: 10.1128/mcb.5.6.1490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thompson C. B., Challoner P. B., Neiman P. E., Groudine M. Levels of c-myc oncogene mRNA are invariant throughout the cell cycle. 1985 Mar 28-Apr 3Nature. 314(6009):363–366. doi: 10.1038/314363a0. [DOI] [PubMed] [Google Scholar]
  52. Wagner M. J., Sharp J. A., Summers W. C. Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1441–1445. doi: 10.1073/pnas.78.3.1441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weissbach A. Eukaryotic DNA polymerases. Annu Rev Biochem. 1977;46:25–47. doi: 10.1146/annurev.bi.46.070177.000325. [DOI] [PubMed] [Google Scholar]
  54. White B. A., Bancroft F. C. Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J Biol Chem. 1982 Aug 10;257(15):8569–8572. [PubMed] [Google Scholar]
  55. Wiedemann L. M., Johnson L. F. Regulation of dihydrofolate reductase synthesis in an overproducing 3T6 cell line during transition from resting to growing state. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2818–2822. doi: 10.1073/pnas.76.6.2818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Willecke K., Teber T., Kucherlapati R. S., Ruddle F. H. Human mitochondrial thymidine kinase is coded for by a gene on chromosome 16 of the nucleus. Somatic Cell Genet. 1977 May;3(3):237–245. doi: 10.1007/BF01538743. [DOI] [PubMed] [Google Scholar]

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