Abstract
We have made Chinese hamster ovary (CHO) cell lines that contain up to 2000 copies of the coding region of the mouse c-myc gene fused to the promoter of the Drosophila gene (hsp70) encoding a Mr 70,000 heat shock protein. Incubation of these cells at 43 degrees C results in an estimated 100-fold induction of c-myc mRNA. Translation of this mRNA occurs when the cells are returned to 37 degrees C, and during the first 3 hr of recovery at 37 degrees C, the c-myc protein is one of the most abundantly synthesized proteins in the cells. The products of the induced c-myc gene are phosphoproteins of apparent Mr 64,000, 66,000, and 75,000. Induced cells die, suggesting that elevated levels of c-myc are cytotoxic. Amplification of genes placed under control of the Drosophila hsp70 promoter may provide a general method for inducibly over expressing proteins in mammalian cells.
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- Alt F. W., Kellems R. E., Bertino J. R., Schimke R. T. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem. 1978 Mar 10;253(5):1357–1370. [PubMed] [Google Scholar]
- Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
- Ballinger D. G., Pardue M. L. The control of protein synthesis during heat shock in Drosophila cells involves altered polypeptide elongation rates. Cell. 1983 May;33(1):103–113. doi: 10.1016/0092-8674(83)90339-2. [DOI] [PubMed] [Google Scholar]
- Berkner K. L., Sharp P. A. Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant. Nucleic Acids Res. 1985 Feb 11;13(3):841–857. doi: 10.1093/nar/13.3.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
- Erisman M. D., Rothberg P. G., Diehl R. E., Morse C. C., Spandorfer J. M., Astrin S. M. Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene. Mol Cell Biol. 1985 Aug;5(8):1969–1976. doi: 10.1128/mcb.5.8.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
- Goustin A. S., Betsholtz C., Pfeifer-Ohlsson S., Persson H., Rydnert J., Bywater M., Holmgren G., Heldin C. H., Westermark B., Ohlsson R. Coexpression of the sis and myc proto-oncogenes in developing human placenta suggests autocrine control of trophoblast growth. Cell. 1985 May;41(1):301–312. doi: 10.1016/0092-8674(85)90083-2. [DOI] [PubMed] [Google Scholar]
- 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]
- Hann S. R., Abrams H. D., Rohrschneider L. R., Eisenman R. N. Proteins encoded by v-myc and c-myc oncogenes: identification and localization in acute leukemia virus transformants and bursal lymphoma cell lines. Cell. 1983 Oct;34(3):789–798. doi: 10.1016/0092-8674(83)90535-4. [DOI] [PubMed] [Google Scholar]
- Hann S. R., Eisenman R. N. Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells. Mol Cell Biol. 1984 Nov;4(11):2486–2497. doi: 10.1128/mcb.4.11.2486. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hayward W. S., Neel B. G., Astrin S. M. Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis. Nature. 1981 Apr 9;290(5806):475–480. doi: 10.1038/290475a0. [DOI] [PubMed] [Google Scholar]
- Hollenberg C. P. Cloning with 2-micrometer DNA vectors and the expression of foreign genes in Saccharomyces cerevisiae. Curr Top Microbiol Immunol. 1982;96:119–144. doi: 10.1007/978-3-642-68315-2_8. [DOI] [PubMed] [Google Scholar]
- Holmgren R., Livak K., Morimoto R., Freund R., Meselson M. Studies of cloned sequences from four Drosophila heat shock loci. Cell. 1979 Dec;18(4):1359–1370. doi: 10.1016/0092-8674(79)90246-0. [DOI] [PubMed] [Google Scholar]
- Kaufman R. J., Sharp P. A. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary dna gene. J Mol Biol. 1982 Aug 25;159(4):601–621. doi: 10.1016/0022-2836(82)90103-6. [DOI] [PubMed] [Google Scholar]
- Kaufman R. J., Wasley L. C., Spiliotes A. J., Gossels S. D., Latt S. A., Larsen G. R., Kay R. M. Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Mol Cell Biol. 1985 Jul;5(7):1750–1759. doi: 10.1128/mcb.5.7.1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kingston R. E., Kaufman R. J., Sharp P. A. Regulation of transcription of the adenovirus EII promoter by EIa gene products: absence of sequence specificity. Mol Cell Biol. 1984 Oct;4(10):1970–1977. doi: 10.1128/mcb.4.10.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18;304(5927):596–602. doi: 10.1038/304596a0. [DOI] [PubMed] [Google Scholar]
- Lau Y. F., Lin C. C., Kan Y. W. Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells. Mol Cell Biol. 1984 Aug;4(8):1469–1475. doi: 10.1128/mcb.4.8.1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leder P., Battey J., Lenoir G., Moulding C., Murphy W., Potter H., Stewart T., Taub R. Translocations among antibody genes in human cancer. Science. 1983 Nov 18;222(4625):765–771. doi: 10.1126/science.6356357. [DOI] [PubMed] [Google Scholar]
- Mayrand S., Pederson T. Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells. Mol Cell Biol. 1983 Feb;3(2):161–171. doi: 10.1128/mcb.3.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miyamoto C., Chizzonite R., Crowl R., Rupprecht K., Kramer R., Schaber M., Kumar G., Poonian M., Ju G. Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7232–7236. doi: 10.1073/pnas.82.21.7232. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miyamoto C., Smith G. E., Farrell-Towt J., Chizzonite R., Summers M. D., Ju G. Production of human c-myc protein in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1985 Oct;5(10):2860–2865. doi: 10.1128/mcb.5.10.2860. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mulligan R. C., Howard B. H., Berg P. Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome. Nature. 1979 Jan 11;277(5692):108–114. doi: 10.1038/277108a0. [DOI] [PubMed] [Google Scholar]
- Persson H., Gray H. E., Godeau F. Growth-dependent synthesis of c-myc-encoded proteins: early stimulation by serum factors in synchronized mouse 3T3 cells. Mol Cell Biol. 1985 Nov;5(11):2903–2912. doi: 10.1128/mcb.5.11.2903. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ramsay G., Evan G. I., Bishop J. M. The protein encoded by the human proto-oncogene c-myc. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7742–7746. doi: 10.1073/pnas.81.24.7742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
- Scahill S. J., Devos R., Van der Heyden J., Fiers W. Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4654–4658. doi: 10.1073/pnas.80.15.4654. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwab M., Ramsay G., Alitalo K., Varmus H. E., Bishop J. M., Martinsson T., Levan G., Levan A. Amplification and enhanced expression of the c-myc oncogene in mouse SEWA tumour cells. Nature. 1985 May 23;315(6017):345–347. doi: 10.1038/315345a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Siebenlist U., Hennighausen L., Battey J., Leder P. Chromatin structure and protein binding in the putative regulatory region of the c-myc gene in Burkitt lymphoma. Cell. 1984 Jun;37(2):381–391. doi: 10.1016/0092-8674(84)90368-4. [DOI] [PubMed] [Google Scholar]
- Smith G. E., Summers M. D., Fraser M. J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983 Dec;3(12):2156–2165. doi: 10.1128/mcb.3.12.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thummel C., Tjian R., Hu S. L., Grodzicker T. Translational control of SV40 T antigen expressed from the adenovirus late promoter. Cell. 1983 Jun;33(2):455–464. doi: 10.1016/0092-8674(83)90427-0. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tuite M. F., Dobson M. J., Roberts N. A., King R. M., Burke D. C., Kingsman S. M., Kingsman A. J. Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae. EMBO J. 1982;1(5):603–608. doi: 10.1002/j.1460-2075.1982.tb01215.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Urlaub G., Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watt R. A., Shatzman A. R., Rosenberg M. Expression and characterization of the human c-myc DNA-binding protein. Mol Cell Biol. 1985 Mar;5(3):448–456. doi: 10.1128/mcb.5.3.448. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Westin E. H., Wong-Staal F., Gelmann E. P., Dalla-Favera R., Papas T. S., Lautenberger J. A., Eva A., Reddy E. P., Tronick S. R., Aaronson S. A. Expression of cellular homologues of retroviral onc genes in human hematopoietic cells. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2490–2494. doi: 10.1073/pnas.79.8.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]