Skip to main content
PMC 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
. 1988 Feb;85(3):718–722. doi: 10.1073/pnas.85.3.718

In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling.

S Hirose 1, Y Suzuki 1
PMCID: PMC279626  PMID: 2829200

Abstract

In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that were required for the supercoiling and for the efficient transcription of the ccc template in the acceptor HeLa cell extract. The 0.6 M KCl fraction had a DNA topoisomerase II activity, and the flow-through fraction contained a supercoiling factor that, with the aid of topoisomerase II, introduced negative supercoils into ccc DNA. When both fractions were added to the posterior silk gland extract, more supercoiling occurred than with the extract alone. Several genes were optimally transcribed under various extents of supercoiling. The fibroin gene and adenovirus 2 major late promoter were fully transcribed as partially supercoiled templates. The sericin gene required more supercoiling for full transcription, whereas no preference for supercoiling was seen with the transcription of hsp70. These results suggest that DNA topology plays a role in the regulation of gene expression.

Full text

PDF
719

Images in this article

719Image
on p.719
720Image
on p.720
720Image
on p.720
721Image
on p.721
721Image
on p.721
721Image
on p.721
722Image
on p.722

Selected References

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

  1. Cockerill P. N., Garrard W. T. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell. 1986 Jan 31;44(2):273–282. doi: 10.1016/0092-8674(86)90761-0. [DOI] [PubMed] [Google Scholar]
  2. Earnshaw W. C., Halligan B., Cooke C. A., Heck M. M., Liu L. F. Topoisomerase II is a structural component of mitotic chromosome scaffolds. J Cell Biol. 1985 May;100(5):1706–1715. doi: 10.1083/jcb.100.5.1706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Glikin G. C., Ruberti I., Worcel A. Chromatin assembly in Xenopus oocytes: in vitro studies. Cell. 1984 May;37(1):33–41. doi: 10.1016/0092-8674(84)90298-8. [DOI] [PubMed] [Google Scholar]
  4. Han S., Udvardy A., Schedl P. Novobiocin blocks the Drosophila heat shock response. J Mol Biol. 1985 May 5;183(1):13–29. doi: 10.1016/0022-2836(85)90277-3. [DOI] [PubMed] [Google Scholar]
  5. Harland R. M., Weintraub H., McKnight S. L. Transcription of DNA injected into Xenopus oocytes is influenced by template topology. Nature. 1983 Mar 3;302(5903):38–43. doi: 10.1038/302038a0. [DOI] [PubMed] [Google Scholar]
  6. Hirose S., Tsuda M., Suzuki Y. Enhanced transcription of fibroin gene in vitro on covalently closed circular templates. J Biol Chem. 1985 Sep 5;260(19):10557–10562. [PubMed] [Google Scholar]
  7. Hsieh T. Purification and properties of type II DNA topoisomerase from embryos of Drosophila melanogaster. Methods Enzymol. 1983;100:161–170. doi: 10.1016/0076-6879(83)00052-x. [DOI] [PubMed] [Google Scholar]
  8. Kmiec E. B., Razvi F., Worcel A. The role of DNA-mediated transfer of TFIIIA in the concerted gyration and differential activation of the Xenopus 5S RNA genes. Cell. 1986 Apr 25;45(2):209–218. doi: 10.1016/0092-8674(86)90385-5. [DOI] [PubMed] [Google Scholar]
  9. Kmiec E. B., Worcel A. The positive transcription factor of the 5S RNA gene induces a 5S DNA-specific gyration in Xenopus oocyte extracts. Cell. 1985 Jul;41(3):945–953. doi: 10.1016/s0092-8674(85)80075-1. [DOI] [PubMed] [Google Scholar]
  10. Larsen A., Weintraub H. An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin. Cell. 1982 Jun;29(2):609–622. doi: 10.1016/0092-8674(82)90177-5. [DOI] [PubMed] [Google Scholar]
  11. Liu L. F. HeLa toposiomerase I. Methods Enzymol. 1983;100:133–137. [PubMed] [Google Scholar]
  12. Luchnik A. N., Bakayev V. V., Zbarsky I. B., Georgiev G. P. Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin. EMBO J. 1982;1(11):1353–1358. doi: 10.1002/j.1460-2075.1982.tb01322.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mirkovitch J., Mirault M. E., Laemmli U. K. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984 Nov;39(1):223–232. doi: 10.1016/0092-8674(84)90208-3. [DOI] [PubMed] [Google Scholar]
  15. Mizushima-Sugano J., Roeder R. G. Cell-type-specific transcription of an immunoglobulin kappa light chain gene in vitro. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8511–8515. doi: 10.1073/pnas.83.22.8511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moran L., Mirault M. E., Tissières A., Lis J., Schedl P., Artavanis-Tsakonas S., Gehring W. J. Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein. Cell. 1979 May;17(1):1–8. doi: 10.1016/0092-8674(79)90289-7. [DOI] [PubMed] [Google Scholar]
  17. Peck L. J., Wang J. C. Energetics of B-to-Z transition in DNA. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6206–6210. doi: 10.1073/pnas.80.20.6206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Petryniak B., Lutter L. C. Topological characterization of the simian virus 40 transcription complex. Cell. 1987 Jan 30;48(2):289–295. doi: 10.1016/0092-8674(87)90432-6. [DOI] [PubMed] [Google Scholar]
  19. Pruitt S. C., Reeder R. H. Effect of topological constraint on transcription of ribosomal DNA in Xenopus oocytes. Comparison of plasmid and endogenous genes. J Mol Biol. 1984 Mar 25;174(1):121–139. doi: 10.1016/0022-2836(84)90368-1. [DOI] [PubMed] [Google Scholar]
  20. Richet E., Abcarian P., Nash H. A. The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination. Cell. 1986 Sep 26;46(7):1011–1021. doi: 10.1016/0092-8674(86)90700-2. [DOI] [PubMed] [Google Scholar]
  21. Ryoji M., Worcel A. Chromatin assembly in Xenopus oocytes: in vivo studies. Cell. 1984 May;37(1):21–32. doi: 10.1016/0092-8674(84)90297-6. [DOI] [PubMed] [Google Scholar]
  22. Sealy L., Cotten M., Chalkley R. Novobiocin inhibits passive chromatin assembly in vitro. EMBO J. 1986 Dec 1;5(12):3305–3311. doi: 10.1002/j.1460-2075.1986.tb04644.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sive H. L., Heintz N., Roeder R. G. Multiple sequence elements are required for maximal in vitro transcription of a human histone H2B gene. Mol Cell Biol. 1986 Oct;6(10):3329–3340. doi: 10.1128/mcb.6.10.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suzuki Y., Tsuda M., Takiya S., Hirose S., Suzuki E., Kameda M., Ninaki O. Tissue-specific transcription enhancement of the fibroin gene characterized by cell-free systems. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9522–9526. doi: 10.1073/pnas.83.24.9522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tsuda M., Suzuki Y. Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5' flanking sequence far upstream. Cell. 1981 Nov;27(1 Pt 2):175–182. doi: 10.1016/0092-8674(81)90371-8. [DOI] [PubMed] [Google Scholar]
  26. Tsuda M., Suzuki Y. Transcription modulation in vitro of the fibroin gene exerted by a 200-base-pair region upstream from the "TATA" box. Proc Natl Acad Sci U S A. 1983 Dec;80(24):7442–7446. doi: 10.1073/pnas.80.24.7442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Udvardy A., Schedl P., Sander M., Hsieh T. S. Novel partitioning of DNA cleavage sites for Drosophila topoisomerase II. Cell. 1985 Apr;40(4):933–941. doi: 10.1016/0092-8674(85)90353-8. [DOI] [PubMed] [Google Scholar]
  28. Villeponteau B., Lundell M., Martinson H. Torsional stress promotes the DNAase I sensitivity of active genes. Cell. 1984 Dec;39(3 Pt 2):469–478. doi: 10.1016/0092-8674(84)90454-9. [DOI] [PubMed] [Google Scholar]
  29. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  30. Weintraub H. Assembly and propagation of repressed and depressed chromosomal states. Cell. 1985 Oct;42(3):705–711. doi: 10.1016/0092-8674(85)90267-3. [DOI] [PubMed] [Google Scholar]
  31. Weintraub H., Cheng P. F., Conrad K. Expression of transfected DNA depends on DNA topology. Cell. 1986 Jul 4;46(1):115–122. doi: 10.1016/0092-8674(86)90865-2. [DOI] [PubMed] [Google Scholar]
  32. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  33. Wolffe A. P., Andrews M. T., Crawford E., Losa R., Brown D. D. Negative supercoiling is not required for 5S RNA transcription in vitro. Cell. 1987 May 8;49(3):301–303. doi: 10.1016/0092-8674(87)90279-0. [DOI] [PubMed] [Google Scholar]
  34. Yang L., Rowe T. C., Nelson E. M., Liu L. F. In vivo mapping of DNA topoisomerase II-specific cleavage sites on SV40 chromatin. Cell. 1985 May;41(1):127–132. doi: 10.1016/0092-8674(85)90067-4. [DOI] [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