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. 1985 Nov 25;13(22):7959–7977. doi: 10.1093/nar/13.22.7959

A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

A Portela, J A Melero, C Martínez, E Domingo, J Ortín
PMCID: PMC322103  PMID: 2933634

Abstract

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.

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Selected References

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

  1. Alexander D. C., McKnight T. D., Williams B. G. A simplified and efficient vector-primer cDNA cloning system. Gene. 1984 Nov;31(1-3):79–89. doi: 10.1016/0378-1119(84)90197-5. [DOI] [PubMed] [Google Scholar]
  2. Basilico C., Gattoni S., Zouzias D., Valle G. D. Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function. Cell. 1979 Jul;17(3):645–659. doi: 10.1016/0092-8674(79)90272-1. [DOI] [PubMed] [Google Scholar]
  3. Beaton A. R., Krug R. M. Synthesis of the templates for influenza virion RNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4682–4686. doi: 10.1073/pnas.81.15.4682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bittner M., Vapnek D. Versatile cloning vectors derived from the runaway-replication plasmid pKN402. Gene. 1981 Dec;15(4):319–329. doi: 10.1016/0378-1119(81)90175-x. [DOI] [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Brady J., Bolen J. B., Radonovich M., Salzman N., Khoury G. Stimulation of simian virus 40 late gene expression by simian virus 40 tumor antigen. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2040–2044. doi: 10.1073/pnas.81.7.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  8. Breathnach R., Harris B. A. Plasmids for the cloning and expression of full-length double-stranded cDNAs under control of the SV40 early or late gene promoter. Nucleic Acids Res. 1983 Oct 25;11(20):7119–7136. doi: 10.1093/nar/11.20.7119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Buetti E., Diggelmann H. Cloned mouse mammary tumor virus DNA is biologically active in transfected mouse cells and its expression is stimulated by glucocorticoid hormones. Cell. 1981 Feb;23(2):335–345. doi: 10.1016/0092-8674(81)90129-x. [DOI] [PubMed] [Google Scholar]
  10. Carter A. D., Felber B. K., Walling M. J., Jubier M. F., Schmidt C. J., Hamer D. H. Duplicated heavy metal control sequences of the mouse metallothionein-I gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7392–7396. doi: 10.1073/pnas.81.23.7392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chou J. Y., Martin R. G. Complementation analysis of simian virus 40 mutants. J Virol. 1974 May;13(5):1101–1109. doi: 10.1128/jvi.13.5.1101-1109.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Desselberger U., Racaniello V. R., Zazra J. J., Palese P. The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity. Gene. 1980 Feb;8(3):315–328. doi: 10.1016/0378-1119(80)90007-4. [DOI] [PubMed] [Google Scholar]
  13. Durnam D. M., Palmiter R. D. Transcriptional regulation of the mouse metallothionein-I gene by heavy metals. J Biol Chem. 1981 Jun 10;256(11):5712–5716. [PubMed] [Google Scholar]
  14. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  15. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  18. Holland J., Spindler K., Horodyski F., Grabau E., Nichol S., VandePol S. Rapid evolution of RNA genomes. Science. 1982 Mar 26;215(4540):1577–1585. doi: 10.1126/science.7041255. [DOI] [PubMed] [Google Scholar]
  19. Jay G., Nomura S., Anderson C. W., Khoury G. Identification of the SV40 agnogene product: a DNA binding protein. Nature. 1981 May 28;291(5813):346–349. doi: 10.1038/291346a0. [DOI] [PubMed] [Google Scholar]
  20. Karin M., Haslinger A., Holtgreve H., Richards R. I., Krauter P., Westphal H. M., Beato M. Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene. Nature. 1984 Apr 5;308(5959):513–519. doi: 10.1038/308513a0. [DOI] [PubMed] [Google Scholar]
  21. Keller J. M., Alwine J. C. Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication. Cell. 1984 Feb;36(2):381–389. doi: 10.1016/0092-8674(84)90231-9. [DOI] [PubMed] [Google Scholar]
  22. Kitamura N., Semler B. L., Rothberg P. G., Larsen G. R., Adler C. J., Dorner A. J., Emini E. A., Hanecak R., Lee J. J., van der Werf S. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature. 1981 Jun 18;291(5816):547–553. doi: 10.1038/291547a0. [DOI] [PubMed] [Google Scholar]
  23. Lai C. J., Nathans D. Mapping temperature-sensitive mutants of simian virus 40: rescue of mutants by fragments of viral DNA. Virology. 1974 Aug;60(2):466–475. doi: 10.1016/0042-6822(74)90340-7. [DOI] [PubMed] [Google Scholar]
  24. Lamb R. A., Lai C. J. Expression of unspliced NS1 mRNA, spliced NS2 mRNA, and a spliced chimera mRNA from cloned influenza virus NS DNA in an SV40 vector. Virology. 1984 May;135(1):139–147. doi: 10.1016/0042-6822(84)90124-7. [DOI] [PubMed] [Google Scholar]
  25. Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  28. Martínez C., del Rio L., Portela A., Domingo E., Ortín J. Evolution of the influenza virus neuraminidase gene during drift of the N2 subtype. Virology. 1983 Oct 30;130(2):539–545. doi: 10.1016/0042-6822(83)90108-3. [DOI] [PubMed] [Google Scholar]
  29. Melero J. A., Gonzalez-Rodriguez J. Preparation of monoclonal antibodies against glycoprotein IIIa of human platelets. Their effect on platelet aggregation. Eur J Biochem. 1984 Jun 1;141(2):421–427. doi: 10.1111/j.1432-1033.1984.tb08208.x. [DOI] [PubMed] [Google Scholar]
  30. Miller J., Bullock P., Botchan M. Simian virus 40 T antigen is required for viral excision from chromosomes. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7534–7538. doi: 10.1073/pnas.81.23.7534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ortin J., Doerfler W. Transcription of the genome of adenovirus type 12. I. Viral mRNA in abortively infected and transformed cells. J Virol. 1975 Jan;15(1):27–35. doi: 10.1128/jvi.15.1.27-35.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ortín J., Martínez C., del Río L., Dávila M., López-Galíndez C., Villanueva N., Domingo E. Evolution of the nucleotide sequence of influenza virus RNA segment 7 during drift of the H3N2 subtype. Gene. 1983 Aug;23(2):233–239. doi: 10.1016/0378-1119(83)90055-0. [DOI] [PubMed] [Google Scholar]
  35. Ortín J., Nájera R., López C., Dávila M., Domingo E. Genetic variability of Hong Kong (H3N2) influenza viruses: spontaneous mutations and their location in the viral genome. Gene. 1980 Nov;11(3-4):319–331. doi: 10.1016/0378-1119(80)90072-4. [DOI] [PubMed] [Google Scholar]
  36. Palese P., Young J. F. Variation of influenza A, B, and C viruses. Science. 1982 Mar 19;215(4539):1468–1474. doi: 10.1126/science.7038875. [DOI] [PubMed] [Google Scholar]
  37. Pavlakis G. N., Hamer D. H. Regulation of a metallothionein-growth hormone hybrid gene in bovine papilloma virus. Proc Natl Acad Sci U S A. 1983 Jan;80(2):397–401. doi: 10.1073/pnas.80.2.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ponta H., Kennedy N., Skroch P., Hynes N. E., Groner B. Hormonal response region in the mouse mammary tumor virus long terminal repeat can be dissociated from the proviral promoter and has enhancer properties. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1020–1024. doi: 10.1073/pnas.82.4.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Racaniello V. R., Baltimore D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4887–4891. doi: 10.1073/pnas.78.8.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Rio D. C., Clark S. G., Tjian R. A mammalian host-vector system that regulates expression and amplification of transfected genes by temperature induction. Science. 1985 Jan 4;227(4682):23–28. doi: 10.1126/science.2981116. [DOI] [PubMed] [Google Scholar]
  42. Rio D. C., Tjian R. Multiple control elements involved in the initiation of SV40 late transcription. J Mol Appl Genet. 1984;2(5):423–435. [PubMed] [Google Scholar]
  43. Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
  44. Stanway G., Hughes P. J., Mountford R. C., Reeve P., Minor P. D., Schild G. C., Almond J. W. Comparison of the complete nucleotide sequences of the genomes of the neurovirulent poliovirus P3/Leon/37 and its attenuated Sabin vaccine derivative P3/Leon 12a1b. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1539–1543. doi: 10.1073/pnas.81.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stuart G. W., Searle P. F., Chen H. Y., Brinster R. L., Palmiter R. D. A 12-base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous gene. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7318–7322. doi: 10.1073/pnas.81.23.7318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Studier F. W. Bacteriophage T7. Science. 1972 Apr 28;176(4033):367–376. doi: 10.1126/science.176.4033.367. [DOI] [PubMed] [Google Scholar]
  47. Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  48. Toyoda H., Kohara M., Kataoka Y., Suganuma T., Omata T., Imura N., Nomoto A. Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants. J Mol Biol. 1984 Apr 25;174(4):561–585. doi: 10.1016/0022-2836(84)90084-6. [DOI] [PubMed] [Google Scholar]
  49. Young J. F., Desselberger U., Palese P., Ferguson B., Shatzman A. R., Rosenberg M. Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Oct;80(19):6105–6109. doi: 10.1073/pnas.80.19.6105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. del Río L., Martínez C., Domingo E., Ortín J. In vitro synthesis of full-length influenza virus complementary RNA. EMBO J. 1985 Jan;4(1):243–247. doi: 10.1002/j.1460-2075.1985.tb02342.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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