Skip to main content
NCBI home page
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
. 1991 May 15;88(10):4513–4517. doi: 10.1073/pnas.88.10.4513

In vitro transcription of baculovirus immediate early genes: accurate mRNA initiation by nuclear extracts from both insect and human cells.

R R Hoopes Jr 1, G F Rohrmann 1
PMCID: PMC51691  PMID: 2034691

Abstract

The production and characterization of nuclear extracts from uninfected Spodoptera frugiperda cells, capable of accurately initiating transcription of baculovirus immediate early genes in vitro, are described. Optimal in vitro transcription was dependent on the presence of a TATA box promoter element and was abolished by alpha-amanitin. Nuclear extracts from the S. frugiperda cells primed with plasmid DNA containing the adenovirus major late promoter produced run-off transcripts of the size predicted for initiation from the adenovirus promoter. In addition, nuclear extracts prepared from a human cell line accurately initiated transcription from the promoter of the baculovirus immediate early gene encoding gp64. Primer extension analysis showed that transcripts derived from the gp64 gene promoter using both the S. frugiperda and human cell nuclear extracts initiated at the same nucleotide as transcripts produced in vivo.

Full text

PDF
4513

Images in this article

4514Image
on p.4514
4515Image
on p.4515
4515Image
on p.4515
4516Image
on p.4516
4516Image
on p.4516
4516Image
on p.4516
4516Image
on p.4516
4516Image
on p.4516
4517Image
on p.4517

Selected References

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

  1. Blissard G. W., Quant-Russell R. L., Rohrmann G. F., Beaudreau G. S. Nucleotide sequence, transcriptional mapping, and temporal expression of the gene encoding p39, a major structural protein of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata. Virology. 1989 Feb;168(2):354–362. doi: 10.1016/0042-6822(89)90276-6. [DOI] [PubMed] [Google Scholar]
  2. Blissard G. W., Rohrmann G. F. Baculovirus diversity and molecular biology. Annu Rev Entomol. 1990;35:127–155. doi: 10.1146/annurev.en.35.010190.001015. [DOI] [PubMed] [Google Scholar]
  3. Blissard G. W., Rohrmann G. F. Location, sequence, transcriptional mapping, and temporal expression of the gp64 envelope glycoprotein gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology. 1989 Jun;170(2):537–555. doi: 10.1016/0042-6822(89)90445-5. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Bucher P. Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. J Mol Biol. 1990 Apr 20;212(4):563–578. doi: 10.1016/0022-2836(90)90223-9. [DOI] [PubMed] [Google Scholar]
  6. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dignam J. D., Martin P. L., Shastry B. S., Roeder R. G. Eukaryotic gene transcription with purified components. Methods Enzymol. 1983;101:582–598. doi: 10.1016/0076-6879(83)01039-3. [DOI] [PubMed] [Google Scholar]
  8. Grula M. A., Buller P. L., Weaver R. F. alpha-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells. J Virol. 1981 Jun;38(3):916–921. doi: 10.1128/jvi.38.3.916-921.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guarino L. A., Summers M. D. Nucleotide sequence and temporal expression of a baculovirus regulatory gene. J Virol. 1987 Jul;61(7):2091–2099. doi: 10.1128/jvi.61.7.2091-2099.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Heiermann R., Pongs O. In vitro transcription with extracts of nuclei of Drosophila embryos. Nucleic Acids Res. 1985 Apr 25;13(8):2709–2730. doi: 10.1093/nar/13.8.2709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirose S., Suzuki Y. In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling. Proc Natl Acad Sci U S A. 1988 Feb;85(3):718–722. doi: 10.1073/pnas.85.3.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  13. Lue N. F., Flanagan P. M., Sugimoto K., Kornberg R. D. Initiation by yeast RNA polymerase II at the adenoviral major late promoter in vitro. Science. 1989 Nov 3;246(4930):661–664. doi: 10.1126/science.2510298. [DOI] [PubMed] [Google Scholar]
  14. Matsuo T., Heller M., Petti L., O'Shiro E., Kieff E. Persistence of the entire Epstein-Barr virus genome integrated into human lymphocyte DNA. Science. 1984 Dec 14;226(4680):1322–1325. doi: 10.1126/science.6095452. [DOI] [PubMed] [Google Scholar]
  15. Pennock G. D., Shoemaker C., Miller L. K. Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector. Mol Cell Biol. 1984 Mar;4(3):399–406. doi: 10.1128/mcb.4.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Penotti F. E. Human DNA TATA boxes and transcription initiation sites. A statistical study. J Mol Biol. 1990 May 5;213(1):37–52. doi: 10.1016/S0022-2836(05)80120-2. [DOI] [PubMed] [Google Scholar]
  17. Rohrmann G. F. Polyhedrin structure. J Gen Virol. 1986 Aug;67(Pt 8):1499–1513. doi: 10.1099/0022-1317-67-8-1499. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sawadogo M., Roeder R. G. Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4394–4398. doi: 10.1073/pnas.82.13.4394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Smale S. T., Baltimore D. The "initiator" as a transcription control element. Cell. 1989 Apr 7;57(1):103–113. doi: 10.1016/0092-8674(89)90176-1. [DOI] [PubMed] [Google Scholar]
  21. Smale S. T., Schmidt M. C., Berk A. J., Baltimore D. Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4509–4513. doi: 10.1073/pnas.87.12.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smith G. E., Summers M. D., Fraser M. J. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol. 1983 Dec;3(12):2156–2165. doi: 10.1128/mcb.3.12.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wiest D. K., Hawley D. K. In vitro analysis of a transcription termination site for RNA polymerase II. Mol Cell Biol. 1990 Nov;10(11):5782–5795. doi: 10.1128/mcb.10.11.5782. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES