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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1973 Aug;70(8):2304–2308. doi: 10.1073/pnas.70.8.2304

In Vitro Transcription of Simian Virus 40 Sequences in SV3T3 Chromatin

Susan M Astrin 1,2
PMCID: PMC433723  PMID: 4365372

Abstract

The template properties of chromatin were studied in a system in which transcription of unique genes could be examined. Chromation and DNA were isolated from SV3T3 cells, a line of simian virus 40 (SV40)-transformed mouse cells, and used as templates for Escherichia coli RNA polymerase. The SV40 sequences in the RNA transcribed in vitro and in RNA isolated from the transformed cells were analyzed by hybridization to radioactive RNA transcribed from SV40 DNA. The results indicate that the same sequences of the SV40 “-” strand (DNA strand which serves as a template for early RNA) are transcribed in vitro from chromatin as are found in transformed cells. In contrast, when the DNA isolated from SV3T3 chromatin by removal of proteins is used as a template, the entire SV40 “-” strand is transcribed. It is concluded that the specific template restriction observed with chromatin is due to the presence of proteins associated with the DNA.

Keywords: transformed mouse cells, hybridization, Escherichia coli RNA polymerase

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Selected References

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

  1. Bonner J., Dahmus M. E., Fambrough D., Huang R. C., Marushige K., Tuan D. Y. The Biology of Isolated Chromatin: Chromosomes, biologically active in the test tube, provide a powerful tool for the study of gene action. Science. 1968 Jan 5;159(3810):47–56. doi: 10.1126/science.159.3810.47. [DOI] [PubMed] [Google Scholar]
  2. Burgess R. R. A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6160–6167. [PubMed] [Google Scholar]
  3. Dawid I. B., Wolstenholme D. R. Ultracentrifuge and electron microscope studies on the structure of mitochondrial DNA. J Mol Biol. 1967 Sep 14;28(2):233–245. doi: 10.1016/s0022-2836(67)80006-8. [DOI] [PubMed] [Google Scholar]
  4. Gelb L. D., Kohne D. E., Martin M. A. Quantitation of Simian virus 40 sequences in African green monkey, mouse and virus-transformed cell genomes. J Mol Biol. 1971 Apr 14;57(1):129–145. doi: 10.1016/0022-2836(71)90123-9. [DOI] [PubMed] [Google Scholar]
  5. Huang R. C., Huang P. C. Effect of protein-bound RNA associated with chick embryo chromatin on template specificity of the chromatin. J Mol Biol. 1969 Jan;39(2):365–378. doi: 10.1016/0022-2836(69)90323-4. [DOI] [PubMed] [Google Scholar]
  6. Lindstrom D. M., Dulbecco R. Strand orientation of simian virus 40 transcription in productively infected cells. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1517–1520. doi: 10.1073/pnas.69.6.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Paul J., Gilmour R. S. Organ-specific restriction of transcription in mammalian chromatin. J Mol Biol. 1968 Jul 14;34(2):305–316. doi: 10.1016/0022-2836(68)90255-6. [DOI] [PubMed] [Google Scholar]
  8. Paul J., Gilmour R. S. Restriction of deoxyribonucleic acid template activity in chromatin is organ-specific. Nature. 1966 Jun 4;210(5040):992–993. doi: 10.1038/210992a0. [DOI] [PubMed] [Google Scholar]
  9. Sambrook J., Sharp P. A., Keller W. Transcription of Simian virus 40. I. Separation of the strands of SV40 DNA and hybridization of the separated strands to RNA extracted from lytically infected and transformed cells. J Mol Biol. 1972 Sep 14;70(1):57–71. doi: 10.1016/0022-2836(72)90163-5. [DOI] [PubMed] [Google Scholar]
  10. Sambrook J., Westphal H., Srinivasan P. R., Dulbecco R. The integrated state of viral DNA in SV40-transformed cells. Proc Natl Acad Sci U S A. 1968 Aug;60(4):1288–1295. doi: 10.1073/pnas.60.4.1288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Seligy V., Miyagi M. Studies of template activity of chromatin isolated from metabolically active and inactive cells. Exp Cell Res. 1969 Nov;58(1):27–34. doi: 10.1016/0014-4827(69)90113-x. [DOI] [PubMed] [Google Scholar]
  12. Smith K. D., Church R. B., McCarthy B. J. Template specificity of isolated chromatin. Biochemistry. 1969 Nov;8(11):4271–4277. doi: 10.1021/bi00839a007. [DOI] [PubMed] [Google Scholar]
  13. Spelsberg T. C., Hnilica L. S., Ansevin A. T. Proteins of chromatin in template restriction. 3. The macromolecules in specific restriction of the chromatin DNA. Biochim Biophys Acta. 1971 Jan 28;228(2):550–562. doi: 10.1016/0005-2787(71)90061-x. [DOI] [PubMed] [Google Scholar]
  14. Strauss J. H., Jr, Kelly R. B., Sinsheimer R. L. Denaturation of RNA with dimethyl sulfoxide. Biopolymers. 1968 Jun;6(6):793–807. doi: 10.1002/bip.1968.360060604. [DOI] [PubMed] [Google Scholar]
  15. Tan C. H., Miyagi M. Specificity of transcription of chromatin in vitro. J Mol Biol. 1970 Jun 28;50(3):641–653. doi: 10.1016/0022-2836(70)90090-2. [DOI] [PubMed] [Google Scholar]
  16. Todaro G. J., Green H. High frequency of SV40 transformation of mouse cell line 3T3. Virology. 1966 Apr;28(4):756–759. doi: 10.1016/0042-6822(66)90261-3. [DOI] [PubMed] [Google Scholar]
  17. Watkins J. F., Dulbecco R. Production of SV40 virus in heterokaryons of transformed and susceptible cells. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1396–1403. doi: 10.1073/pnas.58.4.1396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Weinberg R. A., Ben-Ishai Z., Newbold J. E. Poly A associated with SV40 messenger RNA. Nat New Biol. 1972 Jul 26;238(82):111–113. doi: 10.1038/newbio238111a0. [DOI] [PubMed] [Google Scholar]
  19. Westphal H. SV40 DNA strand selection by Escherichia coli RNA polymerase. J Mol Biol. 1970 Jun 14;50(2):407–420. doi: 10.1016/0022-2836(70)90201-9. [DOI] [PubMed] [Google Scholar]
  20. Zimmerman S. B., Sandeen D. The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal Biochem. 1966 Feb;14(2):269–277. doi: 10.1016/0003-2697(66)90137-0. [DOI] [PubMed] [Google Scholar]

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