Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Dec 1;111(6):2693–2701. doi: 10.1083/jcb.111.6.2693

A cell cycle analysis of growth-related genes expressed during T lymphocyte maturation

PMCID: PMC2116351  PMID: 2277081

Abstract

Fetal liver or bone marrow-derived T lymphocyte precursors undergo extensive, developmentally regulated proliferation in response to inductive signals from the thymic microenvironment. We have used neonatal mouse thymocytes size-separated by centrifugal elutriation to study the cell cycle stage-specific expression of several genes associated with cell proliferation. These include genes involved in the biosynthesis of deoxyribonucleotide precursors, such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and the M1 and M2 subunits of ribonucleotide reductase, as well as c-myc, a cellular oncogene of unknown function. Using nuclear run-on assays, we observed that the transcription rates for these genes, with the exception of TS, are essentially invariant not only throughout the cell cycle in proliferating cells, but also in noncycling (G0) cells. The TS gene showed a transient increase in transcription rate in cells which bordered between a proliferating and nonproliferating status. Studies of an elutriated T cell line, S49.1, yielded similar results, indicating that the process of immortalization has not affected the transcriptional regulation of these genes. Analysis of steady-state mRNA levels using an RNase protection assay demonstrated that the levels of DHFR and TS mRNA accumulate as thymocytes progress through the cell cycle. In contrast, only the M2 subunit of ribonucleotide reductase showed cyclic regulation. Finally, in contrast to cultured cell models, we observed an abrupt fivefold increase in the steady- state level of c-myc mRNA in the transition from G1 to S-phase. We conclude from these studies that the transcriptional regulation of specific genes necessary for cellular proliferation is a minor component of the developmental modulation of the thymocyte cell cycle.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Blanchard J. M., Piechaczyk M., Dani C., Chambard J. C., Franchi A., Pouyssegur J., Jeanteur P. c-myc gene is transcribed at high rate in G0-arrested fibroblasts and is post-transcriptionally regulated in response to growth factors. Nature. 1985 Oct 3;317(6036):443–445. doi: 10.1038/317443a0. [DOI] [PubMed] [Google Scholar]
  2. Boyer P. D., Diamond R. A., Rothenberg E. V. Changes in inducibility of IL-2 receptor alpha-chain and T cell-receptor expression during thymocyte differentiation in the mouse. J Immunol. 1989 Jun 15;142(12):4121–4130. [PubMed] [Google Scholar]
  3. Chang A. C., Nunberg J. H., Kaufman R. J., Erlich H. A., Schimke R. T., Cohen S. N. Phenotypic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature. 1978 Oct 19;275(5681):617–624. doi: 10.1038/275617a0. [DOI] [PubMed] [Google Scholar]
  4. Chien Y., Becker D. M., Lindsten T., Okamura M., Cohen D. I., Davis M. M. A third type of murine T-cell receptor gene. Nature. 1984 Nov 1;312(5989):31–35. doi: 10.1038/312031a0. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Chrysogelos S., Riley D. E., Stein G., Stein J. A human histone H4 gene exhibits cell cycle-dependent changes in chromatin structure that correlate with its expression. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7535–7539. doi: 10.1073/pnas.82.22.7535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Eriksson S., Martin D. W., Jr Ribonucleotide reductase in cultured mouse lymphoma cells. Cell cycle-dependent variation in the activity of subunit protein M2. J Biol Chem. 1981 Sep 25;256(18):9436–9440. [PubMed] [Google Scholar]
  8. Farnham P. J., Abrams J. M., Schimke R. T. Opposite-strand RNAs from the 5' flanking region of the mouse dihydrofolate reductase gene. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3978–3982. doi: 10.1073/pnas.82.12.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Feder J. N., Assaraf Y. G., Seamer L. C., Schimke R. T. The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells. J Biol Chem. 1989 Dec 5;264(34):20583–20590. [PubMed] [Google Scholar]
  10. Feder J., Yen L., Wijsman E., Wang L., Wilkins L., Schroder J., Spurr N., Cann H., Blumenberg M., Cavalli-Sforza L. L. A systematic approach for detecting high-frequency restriction fragment length polymorphisms using large genomic probes. Am J Hum Genet. 1985 Jul;37(4):635–649. [PMC free article] [PubMed] [Google Scholar]
  11. Fowlkes B. J., Pardoll D. M. Molecular and cellular events of T cell development. Adv Immunol. 1989;44:207–264. doi: 10.1016/s0065-2776(08)60643-4. [DOI] [PubMed] [Google Scholar]
  12. Geyer P. K., Johnson L. F. Molecular cloning of DNA sequences complementary to mouse thymidylate synthase messenger RNA. J Biol Chem. 1984 Jun 10;259(11):7206–7211. [PubMed] [Google Scholar]
  13. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  14. Grosschedl R., Baltimore D. Cell-type specificity of immunoglobulin gene expression is regulated by at least three DNA sequence elements. Cell. 1985 Jul;41(3):885–897. doi: 10.1016/s0092-8674(85)80069-6. [DOI] [PubMed] [Google Scholar]
  15. Guidos C. J., Weissman I. L., Adkins B. Intrathymic maturation of murine T lymphocytes from CD8+ precursors. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7542–7546. doi: 10.1073/pnas.86.19.7542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hann S. R., Thompson C. B., Eisenman R. N. c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells. 1985 Mar 28-Apr 3Nature. 314(6009):366–369. doi: 10.1038/314366a0. [DOI] [PubMed] [Google Scholar]
  17. Hiraki D. D., Nomura D., Yokota T., Arai K., Coffman R. L. Isolation and characterization of expressible cDNA clones for mouse Thy-1: a model system for cDNA expression of cell surface proteins. J Immunol. 1986 Jun 1;136(11):4291–4296. [PubMed] [Google Scholar]
  18. Horibata K., Harris A. W. Mouse myelomas and lymphomas in culture. Exp Cell Res. 1970 Apr;60(1):61–77. doi: 10.1016/0014-4827(70)90489-1. [DOI] [PubMed] [Google Scholar]
  19. Imam A. M., Crossley P. H., Jackman A. L., Little P. F. Analysis of thymidylate synthase gene amplification and of mRNA levels in the cell cycle. J Biol Chem. 1987 May 25;262(15):7368–7373. [PubMed] [Google Scholar]
  20. 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]
  21. Kelly K., Cochran B. H., Stiles C. D., Leder P. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell. 1983 Dec;35(3 Pt 2):603–610. doi: 10.1016/0092-8674(83)90092-2. [DOI] [PubMed] [Google Scholar]
  22. Murray A. W., Kirschner M. W. Dominoes and clocks: the union of two views of the cell cycle. Science. 1989 Nov 3;246(4930):614–621. doi: 10.1126/science.2683077. [DOI] [PubMed] [Google Scholar]
  23. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Nepveu A., Levine R. A., Campisi J., Greenberg M. E., Ziff E. B., Marcu K. B. Alternative modes of c-myc regulation in growth factor-stimulated and differentiating cells. Oncogene. 1987;1(3):243–250. [PubMed] [Google Scholar]
  26. Nepveu A., Marcu K. B. Intragenic pausing and anti-sense transcription within the murine c-myc locus. EMBO J. 1986 Nov;5(11):2859–2865. doi: 10.1002/j.1460-2075.1986.tb04580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pearse M., Wu L., Egerton M., Wilson A., Shortman K., Scollay R. A murine early thymocyte developmental sequence is marked by transient expression of the interleukin 2 receptor. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1614–1618. doi: 10.1073/pnas.86.5.1614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rabbitts P. H., Watson J. V., Lamond A., Forster A., Stinson M. A., Evan G., Fischer W., Atherton E., Sheppard R., Rabbitts T. H. Metabolism of c-myc gene products: c-myc mRNA and protein expression in the cell cycle. EMBO J. 1985 Aug;4(8):2009–2015. doi: 10.1002/j.1460-2075.1985.tb03885.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reed J. C., Alpers J. D., Nowell P. C., Hoover R. G. Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3982–3986. doi: 10.1073/pnas.83.11.3982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rothenberg E., Lugo J. P. Differentiation and cell division in the mammalian thymus. Dev Biol. 1985 Nov;112(1):1–17. doi: 10.1016/0012-1606(85)90114-9. [DOI] [PubMed] [Google Scholar]
  31. Scollay R., Smith J., Stauffer V. Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus. Immunol Rev. 1986 Jun;91:129–157. doi: 10.1111/j.1600-065x.1986.tb01487.x. [DOI] [PubMed] [Google Scholar]
  32. Shen-Ong G. L., Keath E. J., Piccoli S. P., Cole M. D. Novel myc oncogene RNA from abortive immunoglobulin-gene recombination in mouse plasmacytomas. Cell. 1982 Dec;31(2 Pt 1):443–452. doi: 10.1016/0092-8674(82)90137-4. [DOI] [PubMed] [Google Scholar]
  33. Tapscott S. J., Davis R. L., Thayer M. J., Cheng P. F., Weintraub H., Lassar A. B. MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. Science. 1988 Oct 21;242(4877):405–411. doi: 10.1126/science.3175662. [DOI] [PubMed] [Google Scholar]
  34. Thelander L., Berg P. Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase. Mol Cell Biol. 1986 Oct;6(10):3433–3442. doi: 10.1128/mcb.6.10.3433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Thompson D. P., Carter G. L., Cory J. G. Changes in messenger RNA levels for the subunits of ribonucleotide reductase during the cell cycle of leukemia L1210 cells. Cancer Commun. 1989;1(4):253–260. [PubMed] [Google Scholar]
  37. Vriz S., Taylor M., Méchali M. Differential expression of two Xenopus c-myc proto-oncogenes during development. EMBO J. 1989 Dec 20;8(13):4091–4097. doi: 10.1002/j.1460-2075.1989.tb08593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zinn K., DiMaio D., Maniatis T. Identification of two distinct regulatory regions adjacent to the human beta-interferon gene. Cell. 1983 Oct;34(3):865–879. doi: 10.1016/0092-8674(83)90544-5. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES