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. 1981 Feb;37(2):683–697. doi: 10.1128/jvi.37.2.683-697.1981

Properties of simian virus 40 small t antigen overproduced in bacteria.

C S Thummel, T L Burgess, R Tjian
PMCID: PMC171056  PMID: 6261013

Abstract

We constructed a series of bacterial plasmids which contained the Escherichia coli lac promoter fused to a simian virus 40 restriction fragment coding for small t antigen. These plasmids expressed different levels of intact viral protein depending on the length of the constructed ribosome binding site. Small t antigen synthesized by the most efficient producer, HP1, constituted 0.5 to 1% of the total cellular protein. On the basis of extensive characterization by immunoprecipitation, gel electrophoresis, isoelectric focusing, tryptic fingerprint analysis, and chromatographic properties, this plasmid-encoded protein was virtually identical to authentic simian virus 40 small t antigen. Partial purification of the HP1-encoded and authentic small t antigens revealed the presence of both monomeric and multimeric forms.

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Selected References

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

  1. BLACK P. H., ROWE W. P., TURNER H. C., HUEBNER R. J. A SPECIFIC COMPLEMENT-FIXING ANTIGEN PRESENT IN SV40 TUMOR AND TRANSFORMED CELLS. Proc Natl Acad Sci U S A. 1963 Dec;50:1148–1156. doi: 10.1073/pnas.50.6.1148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Backman K., Ptashne M. Maximizing gene expression on a plasmid using recombination in vitro. Cell. 1978 Jan;13(1):65–71. doi: 10.1016/0092-8674(78)90138-1. [DOI] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloemendal H. The vertebrate eye lens. Science. 1977 Jul 8;197(4299):127–138. doi: 10.1126/science.877544. [DOI] [PubMed] [Google Scholar]
  5. Bouck N., Beales N., Shenk T., Berg P., di Mayorca G. New region of the simian virus 40 genome required for efficient viral transformation. Proc Natl Acad Sci U S A. 1978 May;75(5):2473–2477. doi: 10.1073/pnas.75.5.2473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chaconas G., Van de Sande J. H., Church R. B. End group labelling of RNA and double stranded DNA by phosphate exchange catalyzed by bacteriophage T4 induced polynucleotide kinase. Biochem Biophys Res Commun. 1975 Oct 6;66(3):962–969. doi: 10.1016/0006-291x(75)90734-2. [DOI] [PubMed] [Google Scholar]
  7. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cowan K., Tegtmeyer P., Anthony D. D. Relationship of replication and transcription of Simian Virus 40 DNA. Proc Natl Acad Sci U S A. 1973 Jul;70(7):1927–1930. doi: 10.1073/pnas.70.7.1927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crawford L. V., Cole C. N., Smith A. E., Paucha E., Tegtmeyer P., Rundell K., Berg P. Organization and expression of early genes of simian virus 40. Proc Natl Acad Sci U S A. 1978 Jan;75(1):117–121. doi: 10.1073/pnas.75.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crawford L. V., O'Farrell P. Z. Effect of alkylation on the physical properties of simian virus 40 T-antigen species. J Virol. 1979 Feb;29(2):587–596. doi: 10.1128/jvi.29.2.587-596.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crawford L. V., Pim D. C., Lane D. P. An immunochemical investigation of SV40 T-antigens. 2. Quantitation of antigens and antibody activities. Virology. 1980 Jan 30;100(2):314–325. doi: 10.1016/0042-6822(80)90522-x. [DOI] [PubMed] [Google Scholar]
  13. Feunteun J., Kress M., Gardes M., Monier R. Viable deletion mutants in the simian virus 40 early region. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4455–4459. doi: 10.1073/pnas.75.9.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fraser T. H., Bruce B. J. Chicken ovalbumin is synthesized and secreted by Escherichia coli. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5936–5940. doi: 10.1073/pnas.75.12.5936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gilbert W., Müller-Hill B. Isolation of the lac repressor. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1891–1898. doi: 10.1073/pnas.56.6.1891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goeddel D. V., Heyneker H. L., Hozumi T., Arentzen R., Itakura K., Yansura D. G., Ross M. J., Miozzari G., Crea R., Seeburg P. H. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature. 1979 Oct 18;281(5732):544–548. doi: 10.1038/281544a0. [DOI] [PubMed] [Google Scholar]
  17. Graessmann A., Graessmann M., Tjian R., Topp W. C. Simian virus 40 small-t protein is required for loss of actin cable networks in rat cells. J Virol. 1980 Mar;33(3):1182–1191. doi: 10.1128/jvi.33.3.1182-1191.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gray H. B., Jr, Ostrander D. A., Hodnett J. L., Legerski R. J., Robberson D. L. Extracellular nucleases of Pseudomonas BAL 31. I. Characterization of single strand-specific deoxyriboendonuclease and double-strand deoxyriboexonuclease activities. Nucleic Acids Res. 1975 Sep;2(9):1459–1492. doi: 10.1093/nar/2.9.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hassell J. A., Lukanidin E., Fey G., Sambrook J. The structure and expression of two defective adenovirus 2/simian virus 40 hybrids. J Mol Biol. 1978 Apr 5;120(2):209–247. doi: 10.1016/0022-2836(78)90065-7. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Johnsrud L. Contacts between Escherichia coli RNA polymerase and a lac operon promoter. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5314–5318. doi: 10.1073/pnas.75.11.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Katz L., Kingsbury D. T., Helinski D. R. Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J Bacteriol. 1973 May;114(2):577–591. doi: 10.1128/jb.114.2.577-591.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  25. Lewis A. M., Jr, Martin R. G. Oncogenicity of simian virus 40 deletion mutants that induce altered 17-kilodalton t-proteins. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4299–4302. doi: 10.1073/pnas.76.9.4299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Linke H. K., Hunter T., Walter G. Structural relationship between the 100,000- and 17,000- molecular-weight T antigens of simian virus 40 (SV40) as deduced by comparison with the SV40-specific proteins coded by the nondefective adenovirus type 2-SV40 hybrid viruses. J Virol. 1979 Jan;29(1):390–394. doi: 10.1128/jvi.29.1.390-394.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martial J. A., Hallewell R. A., Baxter J. D., Goodman H. M. Human growth hormone: complementary DNA cloning and expression in bacteria. Science. 1979 Aug 10;205(4406):602–607. doi: 10.1126/science.377496. [DOI] [PubMed] [Google Scholar]
  28. Martin R. G., Setlow V. P., Edwards C. A., Vembu D. The roles of the simian virus 40 tumor antigens in transformation of Chinese hamster lung cells. Cell. 1979 Jul;17(3):635–643. doi: 10.1016/0092-8674(79)90271-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Mercereau-Puijalon O., Royal A., Cami B., Garapin A., Krust A., Gannon F., Kourilsky P. Synthesis of an ovalbumin-like protein by Escherichia coli K12 harbouring a recombinant plasmid. Nature. 1978 Oct 12;275(5680):505–510. doi: 10.1038/275505a0. [DOI] [PubMed] [Google Scholar]
  31. Müller-Hill B., Crapo L., Gilbert W. Mutants that make more lac repressor. Proc Natl Acad Sci U S A. 1968 Apr;59(4):1259–1264. doi: 10.1073/pnas.59.4.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  33. Paucha E., Mellor A., Harvey R., Smith A. E., Hewick R. M., Waterfield M. D. Large and small tumor antigens from simian virus 40 have identical amino termini mapping at 0.65 map units. Proc Natl Acad Sci U S A. 1978 May;75(5):2165–2169. doi: 10.1073/pnas.75.5.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Prives C., Gilboa E., Revel M., Winocour E. Cell-free translation of simian virus 40 early messenger RNA coding for viral T-antigen. Proc Natl Acad Sci U S A. 1977 Feb;74(2):457–461. doi: 10.1073/pnas.74.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reddy V. B., Thimmappaya B., Dhar R., Subramanian K. N., Zain B. S., Pan J., Ghosh P. K., Celma M. L., Weissman S. M. The genome of simian virus 40. Science. 1978 May 5;200(4341):494–502. doi: 10.1126/science.205947. [DOI] [PubMed] [Google Scholar]
  36. Reed S. I., Stark G. R., Alwine J. C. Autoregulation of simian virus 40 gene A by T antigen. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3083–3087. doi: 10.1073/pnas.73.9.3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Roberts T. M., Bikel I., Yocum R. R., Livingston D. M., Ptashne M. Synthesis of simian virus 40 t antigen in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5596–5600. doi: 10.1073/pnas.76.11.5596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Roberts T. M., Kacich R., Ptashne M. A general method for maximizing the expression of a cloned gene. Proc Natl Acad Sci U S A. 1979 Feb;76(2):760–764. doi: 10.1073/pnas.76.2.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Seif R., Martin R. G. Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells. J Virol. 1979 Dec;32(3):979–988. doi: 10.1128/jvi.32.3.979-988.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sleigh M. J., Topp W. C., Hanich R., Sambrook J. F. Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell. 1978 May;14(1):79–88. doi: 10.1016/0092-8674(78)90303-3. [DOI] [PubMed] [Google Scholar]
  43. Smith A. E., Smith R., Paucha E. Extraction and fingerprint analysis of simian virus 40 large and small T-antigens. J Virol. 1978 Oct;28(1):140–153. doi: 10.1128/jvi.28.1.140-153.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Steinberg B. M., Pollack R. Anchorage independence: analysis of factors affecting the growth and colony formation of wild-type and dl 54/59 mutant SV40-transformed lines. Virology. 1979 Dec;99(2):302–311. doi: 10.1016/0042-6822(79)90009-6. [DOI] [PubMed] [Google Scholar]
  46. Steinberg B., Pollack R., Topp W., Botchan M. Isolation and characterization of T antigen-negative revertants from a line of transformed rat cells containing one copy of the SV40 genome. Cell. 1978 Jan;13(1):19–32. doi: 10.1016/0092-8674(78)90134-4. [DOI] [PubMed] [Google Scholar]
  47. Steitz J. A., Jakes K. How ribosomes select initiator regions in mRNA: base pair formation between the 3' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4734–4738. doi: 10.1073/pnas.72.12.4734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  49. Tegtmeyer P. Function of simian virus 40 gene A in transforming infection. J Virol. 1975 Mar;15(3):613–618. doi: 10.1128/jvi.15.3.613-618.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. doi: 10.1128/jvi.10.4.591-598.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Todaro G. J., Takemoto K. K. "Rescued" SV40: increased transforming efficiency in mouse and human cells. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1031–1037. doi: 10.1073/pnas.62.4.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Topp W. C. Variable defectiveness for lytic growth of the dl 54/59 mutants of simian virus 40. J Virol. 1980 Mar;33(3):1208–1210. doi: 10.1128/jvi.33.3.1208-1210.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]

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