Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Oct;81(19):6004–6008. doi: 10.1073/pnas.81.19.6004

Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.

R R Hirschhorn, P Aller, Z A Yuan, C W Gibson, R Baserga
PMCID: PMC391847  PMID: 6207536

Abstract

A library of double-stranded cDNA was constructed from ts13 cells, a G1-specific temperature-sensitive hamster cell line. The cDNAs, cloned into pBR322, were prepared from poly(A)+ mRNA isolated from ts13 cells 6 hr after serum stimulation at the permissive temperature of 34 degrees C. Differential screening of the library with G1-specific and G0-specific single-stranded cDNA probes prepared from the same cells identified five cDNA clones whose sequences were preferentially expressed in G1. Levels of RNA complementary to these clones were 3- to 6-fold higher in G1 than in other phases of the cell cycle. When ts13 cells were arrested in G1 at the restrictive temperature of 39.6 degrees C, the levels of RNA complementary to p13-2A9 and p13-4F1 were as high as 10 times that found in a resting population, while the expression of sequences complementary to p13-2A8 did not significantly change from levels found in G0. RNA and Southern gel blot analysis suggest that these cell-cycle-specific clones represent either low copy or moderately repetitive gene sequences. Results with another ts mutant of the cell cycle, tsAF8, which is a ts mutant of RNA polymerase II, showed that these cell-cycle-specific sequences have a rapid turnover. The use of G1-specific ts mutants of the cell cycle provides an approach to determine which cell-cycle-dependent genes are most relevant to cell cycle progression.

Full text

PDF
6004

Images in this article

6005Image
on p.6005
6006Image
on p.6006
6006Image
on p.6006
6007Image
on p.6007
6007Image
on p.6007

Selected References

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

  1. Ashihara T., Chang S. D., Baserga R. Constancy of the shift-up point in two temperature-sensitive mammalian cell lines that arrest in G1. J Cell Physiol. 1978 Jul;96(1):15–22. doi: 10.1002/jcp.1040960103. [DOI] [PubMed] [Google Scholar]
  2. Augenlicht L. H., Kobrin D. Cloning and screening of sequences expressed in a mouse colon tumor. Cancer Res. 1982 Mar;42(3):1088–1093. [PubMed] [Google Scholar]
  3. Augenlicht L. H., Kobrin D., Pavlovec A., Royston M. E. Elevated expression of an endogenous retroviral long terminal repeat in a mouse colon tumor. J Biol Chem. 1984 Feb 10;259(3):1842–1847. [PubMed] [Google Scholar]
  4. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brickell P. M., Latchman D. S., Murphy D., Willison K., Rigby P. W. Activation of a Qa/Tla class I major histocompatibility antigen gene is a general feature of oncogenesis in the mouse. Nature. 1983 Dec 22;306(5945):756–760. doi: 10.1038/306756a0. [DOI] [PubMed] [Google Scholar]
  6. Burstin S. J., Meiss H. K., Basilico C. A temperature-sensitive cell cycle mutant of the BHK cell line. J Cell Physiol. 1974 Dec;84(3):397–408. doi: 10.1002/jcp.1040840308. [DOI] [PubMed] [Google Scholar]
  7. Campisi J., Gray H. E., Pardee A. B., Dean M., Sonenshein G. E. Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell. 1984 Feb;36(2):241–247. doi: 10.1016/0092-8674(84)90217-4. [DOI] [PubMed] [Google Scholar]
  8. Cochran B. H., Reffel A. C., Stiles C. D. Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell. 1983 Jul;33(3):939–947. doi: 10.1016/0092-8674(83)90037-5. [DOI] [PubMed] [Google Scholar]
  9. Colwill R. W., Sheinin R. ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4644–4648. doi: 10.1073/pnas.80.15.4644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conrad M. N., Newlon C. S. Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome. Mol Cell Biol. 1983 Jun;3(6):1000–1012. doi: 10.1128/mcb.3.6.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeLisle A. J., Graves R. A., Marzluff W. F., Johnson L. F. Regulation of histone mRNA production and stability in serum-stimulated mouse 3T6 fibroblasts. Mol Cell Biol. 1983 Nov;3(11):1920–1929. doi: 10.1128/mcb.3.11.1920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feinberg R. F., Sun L. H., Ordahl C. P., Frankel F. R. Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences. Proc Natl Acad Sci U S A. 1983 Aug;80(16):5042–5046. doi: 10.1073/pnas.80.16.5042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Floros J., Ashihara T., Baserga R. Characterization of ts13 cells a temperature-sensitive mutant of the G1 phase of the cell cycle. Cell Biol Int Rep. 1978 May;2(3):259–269. doi: 10.1016/0309-1651(78)90006-1. [DOI] [PubMed] [Google Scholar]
  14. Galanti N., Jonak G. J., Soprano K. J., Floros J., Kaczmarek L., Weissman S., Reddy V. B., Tilghman S. M., Baserga R. Characterization and biological activity of cloned simian virus 40 DNA fragments. J Biol Chem. 1981 Jun 25;256(12):6469–6474. [PubMed] [Google Scholar]
  15. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hartwell L. H. Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J Mol Biol. 1971 Jul 14;59(1):183–194. doi: 10.1016/0022-2836(71)90420-7. [DOI] [PubMed] [Google Scholar]
  17. Hillcoat B. L., Swett V., Bertino J. R. Increase of dihydrofolate reductase activity in cultured mammalian cells after exposure to methotrexate. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1632–1637. doi: 10.1073/pnas.58.4.1632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ingles C. J. Temperature-sensitive RNA polymerase II mutations in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):405–409. doi: 10.1073/pnas.75.1.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Ketner G., Kelly T. J., Jr Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1102–1106. doi: 10.1073/pnas.73.4.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. LIEBERMAN I., ABRAMS R., OVE P. Changes in the metabolism of ribonucleic acid preceding the synthesis of deoxyribonucleic acid in mammalian cells cultured from the animal. J Biol Chem. 1963 Jun;238:2141–2149. [PubMed] [Google Scholar]
  23. La Bella F., Brown E. H., Basilico C. Changes in the levels of viral and cellular gene-transcripts in the cell cycle of SV40 transformed mouse cells. J Cell Physiol. 1983 Oct;117(1):62–68. doi: 10.1002/jcp.1041170110. [DOI] [PubMed] [Google Scholar]
  24. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  25. Linzer D. I., Nathans D. Growth-related changes in specific mRNAs of cultured mouse cells. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4271–4275. doi: 10.1073/pnas.80.14.4271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lörincz A. T., Reed S. I. Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes. Nature. 1984 Jan 12;307(5947):183–185. doi: 10.1038/307183a0. [DOI] [PubMed] [Google Scholar]
  27. Ordahl C. P., Kioussis D., Tilghman S. M., Ovitt C. E., Fornwald J. Molecular cloning of developmentally regulated, low-abundance mRNA sequences from embryonic muscle. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4519–4523. doi: 10.1073/pnas.77.8.4519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Plumb M., Stein J., Stein G. Coordinate regulation of multiple histone mRNAs during the cell cycle in HeLa cells. Nucleic Acids Res. 1983 Apr 25;11(8):2391–2410. doi: 10.1093/nar/11.8.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reich N. C., Levine A. J. Growth regulation of a cellular tumour antigen, p53, in nontransformed cells. Nature. 1984 Mar 8;308(5955):199–201. doi: 10.1038/308199a0. [DOI] [PubMed] [Google Scholar]
  30. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Rossini M., Baserga S., Huang C. H., Ingles C. J., Baserga R. Changes in RNA polymerase II in a cell cycle-specific temperature-sensitive mutant of hamster cells. J Cell Physiol. 1980 Apr;103(1):97–103. doi: 10.1002/jcp.1041030114. [DOI] [PubMed] [Google Scholar]
  33. Sarkar S., Glassy M. C., Ferrone S., Jones O. W. Cell cycle and the differential expression of HLA-A,B and HLA-DR antigens on human B lymphoid cells. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7297–7301. doi: 10.1073/pnas.77.12.7297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schutzbank T., Robinson R., Oren M., Levine A. J. SV40 large tumor antigen can regulate some cellular transcripts in a positive fashion. Cell. 1982 Sep;30(2):481–490. doi: 10.1016/0092-8674(82)90245-8. [DOI] [PubMed] [Google Scholar]
  35. Scott M. R., Westphal K. H., Rigby P. W. Activation of mouse genes in transformed cells. Cell. 1983 Sep;34(2):557–567. doi: 10.1016/0092-8674(83)90388-4. [DOI] [PubMed] [Google Scholar]
  36. Shales M., Bergsagel J., Ingles C. J. Defective RNA polymerase II in the G1 specific temperature sensitive hamster cell mutant TsAF8. J Cell Physiol. 1980 Dec;105(3):527–532. doi: 10.1002/jcp.1041050317. [DOI] [PubMed] [Google Scholar]
  37. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  38. Talavera A., Basilico C. Temperature sensitive mutants of BHK cells affected in cell cycle progression. J Cell Physiol. 1977 Sep;92(3):425–436. doi: 10.1002/jcp.1040920310. [DOI] [PubMed] [Google Scholar]
  39. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsuchida N., Uesugi S. Structure and functions of the Kirsten murine sarcoma virus genome: molecular cloning of biologically active Kirsten murine sarcoma virus DNA. J Virol. 1981 May;38(2):720–727. doi: 10.1128/jvi.38.2.720-727.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Waechter D. E., Avignolo C., Freund E., Riggenbach C. M., Mercer W. E., McGuire P. M., Baserga R. Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells. Mol Cell Biochem. 1984;60(1):77–82. doi: 10.1007/BF00226301. [DOI] [PubMed] [Google Scholar]
  42. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES