Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Sep 10;11(17):6003–6020. doi: 10.1093/nar/11.17.6003

Generation of adenovirus by transfection of plasmids.

K L Berkner, P A Sharp
PMCID: PMC326332  PMID: 6310523

Abstract

Biologically active fragments of Adenovirus 5 (Ad5) DNA that span the entire genome have been cloned into plasmids. The covalently attached terminal protein was removed and Eco RI linkers added in a fashion that preserves the Ad5 terminal sequences. When plasmids containing overlapping fragments that represent the entire genome are cotransfected onto 293 cells, infectious virus is obtained. Generation of virus depends upon the release of the 0 or 100 mu Ad5 terminus from pBR322 DNA by Eco RI cleavage. During virus production the modified termini of the transfected fragments are corrected exactly to that of wt viral DNA. The above method for preparing adenovirus recombinants has been used to construct a mutant, Ad5 delta (78.9-84.3), lacking most of the non-essential EIII transcriptional unit. This mutant is phenotypically wild type with respect to burst size and kinetics of growth. Surprisingly, it inhibits wt viral growth upon mixed infections of HeLa or 293 cells, apparently at the level of DNA replication.

Full text

PDF
6008

Images in this article

6011Image
on p.6011
6012Image
on p.6012
6014Image
on p.6014
6015Image
on p.6015
6016Image
on p.6016

Selected References

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

  1. Anderson C. W., Lewis J. B., Baum P. R., Gesteland R. F. Simian virus 40-specific polypeptides in AD2+ ND1- and Ad2+ ND4-infected cells. J Virol. 1976 May;18(2):685–692. doi: 10.1128/jvi.18.2.685-692.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Berkner K. L., Folk W. R. Polynucleotide kinase exchange reaction: quantitave assay for restriction endonuclease-generated 5'-phosphoroyl termini in DNA. J Biol Chem. 1977 May 25;252(10):3176–3184. [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  6. Chow L. T., Broker T. R., Lewis J. B. Complex splicing patterns of RNAs from the early regions of adenovirus-2. J Mol Biol. 1979 Oct 25;134(2):265–303. doi: 10.1016/0022-2836(79)90036-6. [DOI] [PubMed] [Google Scholar]
  7. Englund P. T. Analysis of nucleotide sequences at 3' termini of duplex deoxyribonucleic acid with the use of the T4 deoxyribonucleic acid polymerase. J Biol Chem. 1971 May 25;246(10):3269–3276. [PubMed] [Google Scholar]
  8. Frost E., Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. doi: 10.1016/0042-6822(78)90353-7. [DOI] [PubMed] [Google Scholar]
  9. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Hérissé J., Courtois G., Galibert F. Nucleotide sequence of the EcoRI D fragment of adenovirus 2 genome. Nucleic Acids Res. 1980 May 24;8(10):2173–2192. doi: 10.1093/nar/8.10.2173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hérissé J., Galibert F. Nucleotide sequence of the EcoRI E fragment of adenovirus 2 genome. Nucleic Acids Res. 1981 Mar 11;9(5):1229–1240. doi: 10.1093/nar/9.5.1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones N., Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul;17(3):683–689. doi: 10.1016/0092-8674(79)90275-7. [DOI] [PubMed] [Google Scholar]
  16. Jones N., Shenk T. Isolation of deletion and substitution mutants of adenovirus type 5. Cell. 1978 Jan;13(1):181–188. doi: 10.1016/0092-8674(78)90148-4. [DOI] [PubMed] [Google Scholar]
  17. Kelly T. J., Jr, Lewis A. M., Jr Use of nondefective adenovirus-simian virus 40 hybrids for mapping the simian virus 40 genome. J Virol. 1973 Sep;12(3):643–652. doi: 10.1128/jvi.12.3.643-652.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McClelland M. Purification and characterization of two new modification methylases: MClaI from Caryophanon latum L and MTaqI from Thermus aquaticus YTI. Nucleic Acids Res. 1981 Dec 21;9(24):6795–6804. doi: 10.1093/nar/9.24.6795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Persson H., Jansson M., Philipson L. Synthesis and genomic site for an adenovirus type 2 early glycoprotein. J Mol Biol. 1980 Feb 5;136(4):375–394. doi: 10.1016/0022-2836(80)90396-4. [DOI] [PubMed] [Google Scholar]
  22. Pettersson U., Sambrook J. Amount of viral DNA in the genome of cells transformed by adenovirus type 2. J Mol Biol. 1973 Jan;73(1):125–130. doi: 10.1016/0022-2836(73)90164-2. [DOI] [PubMed] [Google Scholar]
  23. Rekosh D. M., Russell W. C., Bellet A. J., Robinson A. J. Identification of a protein linked to the ends of adenovirus DNA. Cell. 1977 Jun;11(2):283–295. doi: 10.1016/0092-8674(77)90045-9. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Rigby P. W. SV40 and polyoma viruses: their analysis by deoxyribonucleic acid recombination in vitro and their use as vectors in eukaryotic systems. Biochem Soc Symp. 1979;44:89–102. [PubMed] [Google Scholar]
  26. Robinson A. J., Younghusband H. B., Bellett A. J. A circula DNA-protein complex from adenoviruses. Virology. 1973 Nov;56(1):54–69. doi: 10.1016/0042-6822(73)90287-0. [DOI] [PubMed] [Google Scholar]
  27. Sharp P. A., Moore C., Haverty J. L. The infectivity of adenovirus 5 DNA-protein complex. Virology. 1976 Dec;75(2):442–456. doi: 10.1016/0042-6822(76)90042-8. [DOI] [PubMed] [Google Scholar]
  28. Solnick D. Construction of an adenovirus-SV40 recombinant producing SV40 T antigen from an adenovirus late promoter. Cell. 1981 Apr;24(1):135–143. doi: 10.1016/0092-8674(81)90509-2. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Stow N. D. Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis. J Virol. 1981 Jan;37(1):171–180. doi: 10.1128/jvi.37.1.171-180.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tamanoi F., Stillman B. W. Function of adenovirus terminal protein in the initiation of DNA replication. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2221–2225. doi: 10.1073/pnas.79.7.2221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thummel C., Tjian R., Grodzicker T. Expression of SV40 T antigen under control of adenovirus promoters. Cell. 1981 Mar;23(3):825–836. doi: 10.1016/0092-8674(81)90447-5. [DOI] [PubMed] [Google Scholar]
  33. Tolun A., Aleström P., Pettersson U. Sequence of inverted terminal repetitions from different adenoviruses: demonstration of conserved sequences and homology between SA7 termini and SV40 DNA. Cell. 1979 Jul;17(3):705–713. doi: 10.1016/0092-8674(79)90277-0. [DOI] [PubMed] [Google Scholar]
  34. Van Ormondt H., Maat J., De Waard A., Van der Eb A. J. The nucleotide sequence of the transforming HpaI-E fragment of adenovirus type 5 DNA. Gene. 1978 Dec;4(4):309–328. doi: 10.1016/0378-1119(78)90048-3. [DOI] [PubMed] [Google Scholar]
  35. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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 Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES