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
. 1984 Jul;81(13):4051–4054. doi: 10.1073/pnas.81.13.4051

Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells.

R S Gilmour, M Allan, J Paul
PMCID: PMC345366  PMID: 6330734

Abstract

The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6). Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose. A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts. It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo.

Full text

PDF
4051

Images in this article

4053Image
on p.4053
4054Image
on p.4054

Selected References

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

  1. Allan M., Lanyon W. G., Paul J. Multiple origins of transcription in the 4.5 Kb upstream of the epsilon-globin gene. Cell. 1983 Nov;35(1):187–197. doi: 10.1016/0092-8674(83)90221-0. [DOI] [PubMed] [Google Scholar]
  2. Axel R., Cedar H., Felsenfeld G. Synthesis of globin ribonucleic acid from duck-reticulocyte chromatin in vitro. Proc Natl Acad Sci U S A. 1973 Jul;70(7):2029–2032. doi: 10.1073/pnas.70.7.2029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barrett T., Maryanka D., Hamlyn P. H., Gould H. J. Nonhistone proteins control gene expression in reconstituted chromatin. Proc Natl Acad Sci U S A. 1974 Dec;71(12):5057–5061. doi: 10.1073/pnas.71.12.5057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chiu J. F., Tsai Y. H., Sakuma K., Hnilica L. S. Regulation of in vitro mRNA transcription by a fraction of chromosomal proteins. J Biol Chem. 1975 Dec 25;250(24):9431–9433. [PubMed] [Google Scholar]
  5. Dale R. M., Martin E., Livingston D. C., Ward D. C. Direct covalent mercuration of nucleotides and polynucleotides. Biochemistry. 1975 Jun 3;14(11):2447–2457. doi: 10.1021/bi00682a027. [DOI] [PubMed] [Google Scholar]
  6. Giesecke K., Sippel A. E., Nguyen-Huu M. C., Groner B., Hynes N. E., Wurtz T., Schütz G. A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments. Nucleic Acids Res. 1977 Nov;4(11):3943–3958. doi: 10.1093/nar/4.11.3943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gilmour R. S., Paul J. Tissue-specific transcription of the globin gene in isolated chromatin. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3440–3442. doi: 10.1073/pnas.70.12.3440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Konkel D. A., Ingram V. M. Is there specific transcription from isolated chromatin? Nucleic Acids Res. 1978 Apr;5(4):1237–1252. doi: 10.1093/nar/5.4.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  10. Pays E., Donaldson D., Gilmour R. S. Specificity of chromatin transcription in vitro. Anomalies due to RNA-dependent RNA synthesis. Biochim Biophys Acta. 1979 Mar 28;562(1):112–130. doi: 10.1016/0005-2787(79)90131-x. [DOI] [PubMed] [Google Scholar]
  11. Pays E., Gilmour R. S. Specificity of chromatin transcription in vitro. Asymmetric transcription of the globin gene by Escherichia coli RNA polymerase. Biochim Biophys Acta. 1981 May 29;653(3):356–367. doi: 10.1016/0005-2787(81)90192-1. [DOI] [PubMed] [Google Scholar]
  12. Smith M. M., Huang R. C. Transcription in vitro of immunoglobulin kappa light chain genes in isolated mouse myeloma nuclei and chromatin. Proc Natl Acad Sci U S A. 1976 Mar;73(3):775–779. doi: 10.1073/pnas.73.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Steggles A. W., Wilson G. N., Kantor J. A., Picciano D. J., Falvey A. K., Anderson W. F. Cell-free transcription of mammalian chromatin: transcription of globin messenger RNA sequences from bone-marrow chromatin with mammalian RNA polymerase. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1219–1223. doi: 10.1073/pnas.71.4.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Zasloff M., Felsenfeld G. Analysis of in vitro transcription of duck reticulocyte chromatin using mercury-substituted ribonucleoside triphosphates. Biochemistry. 1977 Nov 15;16(23):5135–5145. doi: 10.1021/bi00642a029. [DOI] [PubMed] [Google Scholar]
  15. Zasloff M., Felsenfeld G. Use of mercury-substituted ribonucleoside triphosphates can lead to artefacts in the analysis of in vitro chromatin transcrits. Biochem Biophys Res Commun. 1977 Apr 11;75(3):598–603. doi: 10.1016/0006-291x(77)91514-5. [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