Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Feb;11(2):1023–1029. doi: 10.1128/mcb.11.2.1023

The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells.

Y Li 1, D Li 1, K Osborn 1, L F Johnson 1
PMCID: PMC359772  PMID: 1990264

Abstract

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

Full text

PDF
1023

Images in this article

1025Image
on p.1025
1026Image
on p.1026
1027Image
on p.1027

Selected References

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

  1. Ayusawa D., Shimizu K., Koyama H., Kaneda S., Takeishi K., Seno T. Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. J Mol Biol. 1986 Aug 20;190(4):559–567. doi: 10.1016/0022-2836(86)90241-x. [DOI] [PubMed] [Google Scholar]
  2. Buchman A. R., Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988 Oct;8(10):4395–4405. doi: 10.1128/mcb.8.10.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chung S., Perry R. P. Importance of introns for expression of mouse ribosomal protein gene rpL32. Mol Cell Biol. 1989 May;9(5):2075–2082. doi: 10.1128/mcb.9.5.2075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Conrad A. H. Thymidylate synthetase activity in cultured mammalian cells. J Biol Chem. 1971 Mar 10;246(5):1318–1323. [PubMed] [Google Scholar]
  5. DeWille J. W., Harendza C. J., Jenh C. H., Johnson L. F. Analysis of the multiple 5' and 3' termini of poly(A)+ and poly(A)-deficient thymidylate synthase mRNA in growth-stimulated mouse fibroblasts. J Cell Physiol. 1989 Feb;138(2):358–366. doi: 10.1002/jcp.1041380219. [DOI] [PubMed] [Google Scholar]
  6. DeWille J. W., Jenh C. H., Deng T., Harendza C. J., Johnson L. F. Construction and expression of mouse thymidylate synthase minigenes. J Biol Chem. 1988 Jan 5;263(1):84–91. [PubMed] [Google Scholar]
  7. Deng T. L., Li D. W., Jenh C. H., Johnson L. F. Structure of the gene for mouse thymidylate synthase. Locations of introns and multiple transcriptional start sites. J Biol Chem. 1986 Dec 5;261(34):16000–16005. [PubMed] [Google Scholar]
  8. Deng T. L., Li Y., Johnson L. F. Thymidylate synthase gene expression is stimulated by some (but not all) introns. Nucleic Acids Res. 1989 Jan 25;17(2):645–658. doi: 10.1093/nar/17.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deng T., Li Y., Jolliff K., Johnson L. F. The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites. Mol Cell Biol. 1989 Sep;9(9):4079–4082. doi: 10.1128/mcb.9.9.4079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gillies S. D., Morrison S. L., Oi V. T., Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell. 1983 Jul;33(3):717–728. doi: 10.1016/0092-8674(83)90014-4. [DOI] [PubMed] [Google Scholar]
  11. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gross M. K., Merrill G. F. Regulation of thymidine kinase protein levels during myogenic withdrawal from the cell cycle is independent of mRNA regulation. Nucleic Acids Res. 1988 Dec 23;16(24):11625–11643. doi: 10.1093/nar/16.24.11625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gudas J. M., Knight G. B., Pardee A. B. Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4705–4709. doi: 10.1073/pnas.85.13.4705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamer D. H., Leder P. Splicing and the formation of stable RNA. Cell. 1979 Dec;18(4):1299–1302. doi: 10.1016/0092-8674(79)90240-x. [DOI] [PubMed] [Google Scholar]
  15. Harendza C. J., Johnson L. F. Polyadenylylation signal of the mouse thymidylate synthase gene was created by insertion of an L1 repetitive element downstream of the open reading frame. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2531–2535. doi: 10.1073/pnas.87.7.2531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hernandez N., Lucito R. Elements required for transcription initiation of the human U2 snRNA gene coincide with elements required for snRNA 3' end formation. EMBO J. 1988 Oct;7(10):3125–3134. doi: 10.1002/j.1460-2075.1988.tb03179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hernandez N., Weiner A. M. Formation of the 3' end of U1 snRNA requires compatible snRNA promoter elements. Cell. 1986 Oct 24;47(2):249–258. doi: 10.1016/0092-8674(86)90447-2. [DOI] [PubMed] [Google Scholar]
  18. Jenh C. H., Deng T. L., Li D. W., DeWille J., Johnson L. F. Mouse thymidylate synthase messenger RNA lacks a 3' untranslated region. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8482–8486. doi: 10.1073/pnas.83.22.8482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jenh C. H., Geyer P. K., Johnson L. F. Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line. Mol Cell Biol. 1985 Oct;5(10):2527–2532. doi: 10.1128/mcb.5.10.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim Y. K., Wells S., Lau Y. F., Lee A. S. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5894–5898. doi: 10.1073/pnas.85.16.5894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li D. W., Johnson L. F. A mouse thymidylate synthase pseudogene derived from an aberrantly processed RNA molecule. Gene. 1989 Oct 30;82(2):363–370. doi: 10.1016/0378-1119(89)90064-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Nussbaum R. L., Walmsley R. M., Lesko J. G., Airhart S. D., Ledbetter D. H. Thymidylate synthase-deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression. Am J Hum Genet. 1985 Nov;37(6):1192–1205. [PMC free article] [PubMed] [Google Scholar]
  27. Perryman S. M., Rossana C., Deng T. L., Vanin E. F., Johnson L. F. Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme. Mol Biol Evol. 1986 Jul;3(4):313–321. doi: 10.1093/oxfordjournals.molbev.a040400. [DOI] [PubMed] [Google Scholar]
  28. Pfarr D. S., Sathe G., Reff M. E. A highly modular cloning vector for the analysis of eukaryotic genes and gene regulatory elements. DNA. 1985 Dec;4(6):461–467. doi: 10.1089/dna.1985.4.461. [DOI] [PubMed] [Google Scholar]
  29. Rossana C., Gollakota Rao L., Johnson L. F. Thymidylate synthetase overproduction in 5-fluorodeoxyuridine-resistant mouse fibroblasts. Mol Cell Biol. 1982 Sep;2(9):1118–1125. doi: 10.1128/mcb.2.9.1118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rossi P., de Crombrugghe B. Identification of a cell-specific transcriptional enhancer in the first intron of the mouse alpha 2 (type I) collagen gene. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5590–5594. doi: 10.1073/pnas.84.16.5590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sherley J. L., Kelly T. J. Regulation of human thymidine kinase during the cell cycle. J Biol Chem. 1988 Jun 15;263(17):8350–8358. [PubMed] [Google Scholar]
  32. Sisodia S. S., Sollner-Webb B., Cleveland D. W. Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol Cell Biol. 1987 Oct;7(10):3602–3612. doi: 10.1128/mcb.7.10.3602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. TODARO G. J., GREEN H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol. 1963 May;17:299–313. doi: 10.1083/jcb.17.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Travali S., Lipson K. E., Jaskulski D., Lauret E., Baserga R. Role of the promoter in the regulation of the thymidine kinase gene. Mol Cell Biol. 1988 Apr;8(4):1551–1557. doi: 10.1128/mcb.8.4.1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. de Vegvar H. E., Lund E., Dahlberg J. E. 3' end formation of U1 snRNA precursors is coupled to transcription from snRNA promoters. Cell. 1986 Oct 24;47(2):259–266. doi: 10.1016/0092-8674(86)90448-4. [DOI] [PubMed] [Google Scholar]

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

RESOURCES