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. 1979 Jan;76(1):160–164. doi: 10.1073/pnas.76.1.160

RNA synthesis in isolated nuclei: in vitro initiation of adenovirus 2 major late mRNA precursor.

J L Manley, P A Sharp, M L Gefter
PMCID: PMC382896  PMID: 284328

Abstract

We have analyzed the RNA produced in vitro by incubating nuclei from HeLa cells infected with adenovirus serotype 2. Our results show that adenovirus-specific RNA is produced at a linear rate for up to 2.5 hr. Hybridization analysis of RNA produced in nuclei isolated 18 hr after infection indicated that transcription begins at the "late promoter" at map position 16.5. Sequence analysis of the 5' termini of the in vitro transcripts showed that this system initiates RNA chains de novo at the correct promoter and that the 5' terminus is capped.

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Selected References

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  1. Berget S. M., Moore C., Sharp P. A. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3171–3175. doi: 10.1073/pnas.74.8.3171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chow L. T., Broker T. R. The spliced structures of adenovirus 2 fiber message and the other late mRNAs. Cell. 1978 Oct;15(2):497–510. doi: 10.1016/0092-8674(78)90019-3. [DOI] [PubMed] [Google Scholar]
  5. Furuichi Y. "Pretranscriptional capping" in the biosynthesis of cytoplasmic polyhedrosis virus mRNA. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1086–1090. doi: 10.1073/pnas.75.3.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Goldberg S., Weber J., Darnell J. E., Jr The definition of a large viral transcription unit late in Ad2 infection of HeLa cells: mapping by effects of ultraviolet irradiation. Cell. 1977 Apr;10(4):617–621. doi: 10.1016/0092-8674(77)90094-0. [DOI] [PubMed] [Google Scholar]
  7. Klessig D. F. Two adenovirus mRNAs have a common 5' terminal leader sequence encoded at least 10 kb upstream from their main coding regions. Cell. 1977 Sep;12(1):9–21. doi: 10.1016/0092-8674(77)90181-7. [DOI] [PubMed] [Google Scholar]
  8. Lewis J. B., Atkins J. F., Anderson C. W., Baum P. R., Gesteland R. F. Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1344–1348. doi: 10.1073/pnas.72.4.1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Marzluff W. F., Jr, Murphy E. C., Jr, Huang R. C. Transcription of ribonucleic acid in isolated mouse myeloma nuclei. Biochemistry. 1973 Aug 28;12(18):3440–3446. doi: 10.1021/bi00742a013. [DOI] [PubMed] [Google Scholar]
  10. Mory Y. Y., Gefter M. L. Synthesis of messenger RNA-like molecules in isolated myeloma nuclei. Nucleic Acids Res. 1977 Jun;4(6):1739–1757. doi: 10.1093/nar/4.6.1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Penman S. RNA metabolism in the HeLa cell nucleus. J Mol Biol. 1966 May;17(1):117–130. doi: 10.1016/s0022-2836(66)80098-0. [DOI] [PubMed] [Google Scholar]
  12. Piña M., Green M. Biochemical studies on adenovirus multiplication. IX. Chemical and base composition analysis of 28 human adenoviruses. Proc Natl Acad Sci U S A. 1965 Aug;54(2):547–551. doi: 10.1073/pnas.54.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schibler U., Perry R. P. The 5'-termini of heterogeneous nuclear RNA: a comparison among molecules of different sizes and ages. Nucleic Acids Res. 1977 Dec;4(12):4133–4149. doi: 10.1093/nar/4.12.4133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Smith M. M., Reeve A. E., Huang R. C. Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes. Cell. 1978 Oct;15(2):615–626. doi: 10.1016/0092-8674(78)90030-2. [DOI] [PubMed] [Google Scholar]
  15. Vennström B., Pettersson U., Philipson L. Initiation of transcription in nuclei isolated from adenovirus infected cells. Nucleic Acids Res. 1978 Jan;5(1):205–219. doi: 10.1093/nar/5.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Vennström B., Philipson L. Fidelity of adenovirus RNA transcription in isolated HeLa cell nuclei. J Virol. 1977 May;22(2):290–299. doi: 10.1128/jvi.22.2.290-299.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wei C., Moss B. 5'-Terminal capping of RNA by guanylyltransferase from HeLa cell nuclei. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3758–3761. doi: 10.1073/pnas.74.9.3758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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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