Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Mar;13(3):1565–1571. doi: 10.1128/mcb.13.3.1565

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

J Ash 1, Y Ke 1, M Korb 1, L F Johnson 1
PMCID: PMC359468  PMID: 8095091

Abstract

The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.

Full text

PDF
1565

Images in this article

1567Image
on p.1567
1568Image
on p.1568
1568Image
on p.1568
1569Image
on p.1569

Selected References

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

  1. Alder H., Yoshinouchi M., Prystowsky M. B., Appasamy P., Baserga R. A conserved region in intron 1 negatively regulates the expression of the PCNA gene. Nucleic Acids Res. 1992 Apr 11;20(7):1769–1775. doi: 10.1093/nar/20.7.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. Goto K., Okada T. S., Kondoh H. Functional cooperation of lens-specific and nonspecific elements in the delta 1-crystallin enhancer. Mol Cell Biol. 1990 Mar;10(3):958–964. doi: 10.1128/mcb.10.3.958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Gudas J. M., Knight G. B., Pardee A. B. Ordered splicing of thymidine kinase pre-mRNA during the S phase of the cell cycle. Mol Cell Biol. 1990 Oct;10(10):5591–5595. doi: 10.1128/mcb.10.10.5591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Huang M. T., Gorman C. M. Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 1990 Feb 25;18(4):937–947. doi: 10.1093/nar/18.4.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Johnson L. F. G1 events and the regulation of genes for S-phase enzymes. Curr Opin Cell Biol. 1992 Apr;4(2):149–154. doi: 10.1016/0955-0674(92)90025-8. [DOI] [PubMed] [Google Scholar]
  20. Konieczny S. F., Emerson C. P., Jr Complex regulation of the muscle-specific contractile protein (troponin I) gene. Mol Cell Biol. 1987 Sep;7(9):3065–3075. doi: 10.1128/mcb.7.9.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li Y., Li D., Osborn K., Johnson L. F. The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells. Mol Cell Biol. 1991 Feb;11(2):1023–1029. doi: 10.1128/mcb.11.2.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neuberger M. S., Williams G. T. The intron requirement for immunoglobulin gene expression is dependent upon the promoter. Nucleic Acids Res. 1988 Jul 25;16(14B):6713–6724. doi: 10.1093/nar/16.14.6713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Niwa M., Rose S. D., Berget S. M. In vitro polyadenylation is stimulated by the presence of an upstream intron. Genes Dev. 1990 Sep;4(9):1552–1559. doi: 10.1101/gad.4.9.1552. [DOI] [PubMed] [Google Scholar]
  24. Oshima R. G., Abrams L., Kulesh D. Activation of an intron enhancer within the keratin 18 gene by expression of c-fos and c-jun in undifferentiated F9 embryonal carcinoma cells. Genes Dev. 1990 May;4(5):835–848. doi: 10.1101/gad.4.5.835. [DOI] [PubMed] [Google Scholar]
  25. Ottavio L., Chang C. D., Rizzo M. G., Travali S., Casadevall C., Baserga R. Importance of introns in the growth regulation of mRNA levels of the proliferating cell nuclear antigen gene. Mol Cell Biol. 1990 Jan;10(1):303–309. doi: 10.1128/mcb.10.1.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pandey N. B., Chodchoy N., Liu T. J., Marzluff W. F. Introns in histone genes alter the distribution of 3' ends. Nucleic Acids Res. 1990 Jun 11;18(11):3161–3170. doi: 10.1093/nar/18.11.3161. [DOI] [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. 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]
  29. 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]
  30. Smale S. T., Tjian R. Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters. Mol Cell Biol. 1985 Feb;5(2):352–362. doi: 10.1128/mcb.5.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Takayanagi A., Kaneda S., Ayusawa D., Seno T. Intron 1 and the 5'-flanking region of the human thymidylate synthase gene as a regulatory determinant of growth-dependent expression. Nucleic Acids Res. 1992 Aug 11;20(15):4021–4025. doi: 10.1093/nar/20.15.4021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Woychik R. P., Camper S. A., Lyons R. H., Horowitz S., Goodwin E. C., Rottman F. M. Cloning and nucleotide sequencing of the bovine growth hormone gene. Nucleic Acids Res. 1982 Nov 25;10(22):7197–7210. doi: 10.1093/nar/10.22.7197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. de la Peña P., Zasloff M. Enhancement of mRNA nuclear transport by promoter elements. Cell. 1987 Aug 14;50(4):613–619. doi: 10.1016/0092-8674(87)90034-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES