Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Feb 11;11(3):687–706. doi: 10.1093/nar/11.3.687

Constitutive, long-term production of human interferons by hamster cells containing multiple copies of a cloned interferon gene.

J Haynes, C Weissmann
PMCID: PMC325746  PMID: 6188107

Abstract

Hybrid plasmids containing the mouse dihydrofolate reductase (dhfr) and a human interferon (either IFN-alpha 5 or IFN-gamma) coding sequence under the control of viral promoters were transfected into dhfr- Chinese hamster ovary (CHO) cells. dhfr+ colonies produced IFN at 10-1000 units X ml-1 X day-1. Clones selected in methotrexate had a 20-50-fold increase in the IFN-alpha 5 and dhfr DNA and mRNA content and secreted IFN at 20,000-100,000 units X ml-1 X day-1. SDS-polyacrylamide gel electrophoresis of partially purified 35S-HuIFN-gamma from CHO cells showed a multiple of labeled bands with a mobility corresponding to 22,400 to 23,400 daltons which was absent in the supernatants of non-transformed CHO cells. The higher apparent molecular weight of human IFN-gamma from CHO cells as compared to that of human IFN-gamma from E. coli (about 18,800) suggests that the former was glycosylated.

Full text

PDF
687

Images in this article

696Image
on p.696
701Image
on p.701

Selected References

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

  1. Allen G., Fantes K. H. A family of structural genes for human lymphoblastoid (leukocyte-type) interferon. Nature. 1980 Oct 2;287(5781):408–411. doi: 10.1038/287408a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  6. Brack C., Nagata S., Mantei N., Weissmann C. Molecular analysis of the human interferon-alpha gene family. Gene. 1981 Dec;15(4):379–394. doi: 10.1016/0378-1119(81)90181-5. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chasin L. A., Urlaub G. Mutant alleles for hypoxanthine phosphoriboxyltransferase: codominant expression, complementation, and segregation in hybrid Chinese hamster cells. Somatic Cell Genet. 1976 Sep;2(5):453–467. doi: 10.1007/BF01542725. [DOI] [PubMed] [Google Scholar]
  9. Derynck R., Content J., DeClercq E., Volckaert G., Tavernier J., Devos R., Fiers W. Isolation and structure of a human fibroblast interferon gene. Nature. 1980 Jun 19;285(5766):542–547. doi: 10.1038/285542a0. [DOI] [PubMed] [Google Scholar]
  10. Derynck R., Remaut E., Saman E., Stanssens P., De Clercq E., Content J., Fiers W. Expression of human fibroblast interferon gene in Escherichia coli. Nature. 1980 Sep 18;287(5779):193–197. doi: 10.1038/287193a0. [DOI] [PubMed] [Google Scholar]
  11. Fromm M., Berg P. Deletion mapping of DNA regions required for SV40 early region promoter function in vivo. J Mol Appl Genet. 1982;1(5):457–481. [PubMed] [Google Scholar]
  12. Fujisawa J. U., Kawade Y. Properties of nonglycosylated and glycosidase-treated mouse L cell interferon species. Virology. 1981 Jul 30;112(2):480–486. doi: 10.1016/0042-6822(81)90295-6. [DOI] [PubMed] [Google Scholar]
  13. Fujisawa J., Iwakura Y., Kawade Y. Nonglycosylated mouse L cell interferon produced by the action of tunicamycin. J Biol Chem. 1978 Dec 25;253(24):8677–8679. [PubMed] [Google Scholar]
  14. Goeddel D. V., Leung D. W., Dull T. J., Gross M., Lawn R. M., McCandliss R., Seeburg P. H., Ullrich A., Yelverton E., Gray P. W. The structure of eight distinct cloned human leukocyte interferon cDNAs. Nature. 1981 Mar 5;290(5801):20–26. doi: 10.1038/290020a0. [DOI] [PubMed] [Google Scholar]
  15. Goeddel D. V., Yelverton E., Ullrich A., Heyneker H. L., Miozzari G., Holmes W., Seeburg P. H., Dull T., May L., Stebbing N. Human leukocyte interferon produced by E. coli is biologically active. Nature. 1980 Oct 2;287(5781):411–416. doi: 10.1038/287411a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Gray P. W., Leung D. W., Pennica D., Yelverton E., Najarian R., Simonsen C. C., Derynck R., Sherwood P. J., Wallace D. M., Berger S. L. Expression of human immune interferon cDNA in E. coli and monkey cells. Nature. 1982 Feb 11;295(5849):503–508. doi: 10.1038/295503a0. [DOI] [PubMed] [Google Scholar]
  18. Havell E. A., Yamazaki S., Vilcek J. Altered molecular species of human interferon produced in the presence of inhibitors of glycosylation. J Biol Chem. 1977 Jun 25;252(12):4425–4427. [PubMed] [Google Scholar]
  19. Houghton M., Jackson I. J., Porter A. G., Doel S. M., Catlin G. H., Barber C., Carey N. H. The absence of introns within a human fibroblast interferon gene. Nucleic Acids Res. 1981 Jan 24;9(2):247–266. doi: 10.1093/nar/9.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Knight E., Jr Interferon: purification and initial characterization from human diploid cells. Proc Natl Acad Sci U S A. 1976 Feb;73(2):520–523. doi: 10.1073/pnas.73.2.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Langford M. P., Stanton G. J., Johnson H. M. Biological effects of staphylococcal enterotoxin A on human peripheral lymphocytes. Infect Immun. 1978 Oct;22(1):62–68. doi: 10.1128/iai.22.1.62-68.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lawn R. M., Adelman J., Franke A. E., Houck C. M., Gross M., Najarian R., Goeddel D. V. Human fibroblast interferon gene lacks introns. Nucleic Acids Res. 1981 Mar 11;9(5):1045–1052. doi: 10.1093/nar/9.5.1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lin L. S., Wiranowska-Stewart M., Chudzio T., Stewart W. E., 2nd Characterization of the heterogeneous molecules of human interferons: differences in the cross-species antiviral activities of various molecular populations in human leukocyte interferons. J Gen Virol. 1978 Apr;39(1):125–130. doi: 10.1099/0022-1317-39-1-125. [DOI] [PubMed] [Google Scholar]
  26. Masucci M. G., Szigeti R., Klein E., Klein G., Gruest J., Montagnier L., Taira H., Hall A., Nagata S., Weissmann C. Effect of interferon-alpha 1 from E. coli on some cell functions. Science. 1980 Sep 19;209(4463):1431–1435. doi: 10.1126/science.6158096. [DOI] [PubMed] [Google Scholar]
  27. Nagata S., Mantei N., Weissmann C. The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha. Nature. 1980 Oct 2;287(5781):401–408. doi: 10.1038/287401a0. [DOI] [PubMed] [Google Scholar]
  28. Nagata S., Taira H., Hall A., Johnsrud L., Streuli M., Ecsödi J., Boll W., Cantell K., Weissmann C. Synthesis in E. coli of a polypeptide with human leukocyte interferon activity. Nature. 1980 Mar 27;284(5754):316–320. doi: 10.1038/284316a0. [DOI] [PubMed] [Google Scholar]
  29. Noel D., Nikaido K., Ames G. F. A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry. 1979 Sep 18;18(19):4159–4165. doi: 10.1021/bi00586a017. [DOI] [PubMed] [Google Scholar]
  30. O'Hare K., Benoist C., Breathnach R. Transformation of mouse fibroblasts to methotrexate resistance by a recombinant plasmid expressing a prokaryotic dihydrofolate reductase. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1527–1531. doi: 10.1073/pnas.78.3.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ohno S., Taniguchi T. Structure of a chromosomal gene for human interferon beta. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5305–5309. doi: 10.1073/pnas.78.9.5305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Parsons J. T., Coffin J. M., Haroz R. K., Bromley P. A., Weissmann C. Quantitative determination and location of newly synthesized virus-specific ribonucleic acid in chicken cells infected with Rous sarcoma virus. J Virol. 1973 May;11(5):761–774. doi: 10.1128/jvi.11.5.761-774.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Reddy V. B., Ghosh P. K., Lebowitz P., Piatak M., Weissman S. M. Simian virus 40 early mRNA's. I. Genomic localization of 3' and 5' termini and two major splices in mRNA from transformed and lytically infected cells. J Virol. 1979 Apr;30(1):279–296. doi: 10.1128/jvi.30.1.279-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Ringold G., Dieckmann B., Lee F. Co-expression and amplification of dihydrofolate reductase cDNA and the Escherichia coli XGPRT gene in Chinese hamster ovary cells. J Mol Appl Genet. 1981;1(3):165–175. [PubMed] [Google Scholar]
  36. Schellekens H., de Reus A., Bolhuis R., Fountoulakis M., Schein C., Ecsödi J., Nagata S., Weissmann C. Comparative antiviral efficiency of leukocyte and bacterially produced human alpha-interferon in rhesus monkeys. Nature. 1981 Aug 20;292(5825):775–776. doi: 10.1038/292775a0. [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. Strander H., Mogensen K. E., Cantell K. Production of human lymphoblastoid interferon. J Clin Microbiol. 1975 Jan;1(1):116–117. doi: 10.1128/jcm.1.1.116-117.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tan Y. H., Barakat F., Berthold W., Smith-Johannsen H., Tan C. The isolation and amino acid/sugar composition of human fibroblastoid interferon. J Biol Chem. 1979 Aug 25;254(16):8067–8073. [PubMed] [Google Scholar]
  40. Taniguchi T., Guarente L., Roberts T. M., Kimelman D., Douhan J., 3rd, Ptashne M. Expression of the human fibroblast interferon gene in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5230–5233. doi: 10.1073/pnas.77.9.5230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  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]
  43. Weaver R. F., Weissmann C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. Nucleic Acids Res. 1979 Nov 10;7(5):1175–1193. doi: 10.1093/nar/7.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weissmann C., Nagata S., Boll W., Fountoulakis M., Fujisawa A., Fujisawa J. I., Haynes J., Henco K., Mantei N., Ragg H. Structure and expression of human IFN-alpha genes. Philos Trans R Soc Lond B Biol Sci. 1982 Sep 24;299(1094):7–28. doi: 10.1098/rstb.1982.0102. [DOI] [PubMed] [Google Scholar]
  45. Wetzel R. Assignment of the disulphide bonds of leukocyte interferon. Nature. 1981 Feb 12;289(5798):606–607. doi: 10.1038/289606a0. [DOI] [PubMed] [Google Scholar]
  46. Wieslander L. A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem. 1979 Oct 1;98(2):305–309. doi: 10.1016/0003-2697(79)90145-3. [DOI] [PubMed] [Google Scholar]
  47. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  48. Yip Y. K., Barrowclough B. S., Urban C., Vilcek J. Molecular weight of human gamma interferon is similar to that of other human interferons. Science. 1982 Jan 22;215(4531):411–413. doi: 10.1126/science.6173921. [DOI] [PubMed] [Google Scholar]
  49. Yip Y. K., Barrowclough B. S., Urban C., Vilcek J. Purification of two subspecies of human gamma (immune) interferon. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1820–1824. doi: 10.1073/pnas.79.6.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yip Y. K., Pang R. H., Urban C., Vilcek J. Partial purification and characterization of human gamma (immune) interferon. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1601–1605. doi: 10.1073/pnas.78.3.1601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. de Jong W. W., Zweers A., Cohen L. H. Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes. Biochem Biophys Res Commun. 1978 May 30;82(2):532–539. doi: 10.1016/0006-291x(78)90907-5. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES