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. 1979 Jan;6(1):371–383. doi: 10.1093/nar/6.1.371

The specificity of in vitro chromatin transcription.

G F Crouse, E J Fodor, P Doty
PMCID: PMC327694  PMID: 370786

Abstract

The in vitro transcription of chicken reticulocyte chromatin with E. coli RNA polymerase has been studied in several different ways. The amount of globin RNA sequences has been measured by hybridizing the transcript with globin cDNA; we show that under the proper conditions mercurated transcript RNA can be separated from endogenous RNA on sulfhydryl affinity columns. The amount of globin RNA in the transcript is approximately 20 fold greater than that from erythrocyte chromatin or reticulocyte DNA. Although these data could be used to support the hypothesis of specific transcription, we show by RNA/RNA self hybridization of the transcript (which is at least 50% symmetric) and by hybridization of the transcript to unique DNA in vast RNA excess that the bulk of the chromatin transcript differs little from the transcript of naked DNA. Several explanations for these apparently contradictory results are offered with the most likely one being compatible with random transcription of at least most of the sequences in the chromatin.

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Selected References

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

  1. 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]
  2. Axel R., Cedar H., Felsenfield G. The structure of the globin genes in chromatin. Biochemistry. 1975 Jun 3;14(11):2489–2495. doi: 10.1021/bi00682a031. [DOI] [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. Biessmann H., Gjerset R. A., Levy B., McCarthy B. J. Fidelity of chromatin transcription in vitro. Biochemistry. 1976 Oct 5;15(20):4356–4363. doi: 10.1021/bi00665a002. [DOI] [PubMed] [Google Scholar]
  5. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  6. Cedar H., Felsenfeld G. Transcription of chromatin in vitro. J Mol Biol. 1973 Jun 25;77(2):237–254. doi: 10.1016/0022-2836(73)90334-3. [DOI] [PubMed] [Google Scholar]
  7. Crouse G. F., Fodor J. B., Doty P. In vitro transcription of chromatin in the presence of a mercurated nucleotide. Proc Natl Acad Sci U S A. 1976 May;73(5):1564–1567. doi: 10.1073/pnas.73.5.1564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Di Nocera P. P., Avitabile A., Blasi F. In vitro transcription of the Escherichia coli histidine operon primed by dinucleotides. Effect of the first histidine biosynthetic enzyme. J Biol Chem. 1975 Nov 10;250(21):8376–8381. [PubMed] [Google Scholar]
  9. Downey K. M., Jurmark B. S., So A. G. Determination of nucleotide sequences at promoter regions by the use of dinucleotides. Biochemistry. 1971 Dec 21;10(26):4970–4975. doi: 10.1021/bi00802a021. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Gilmour R. S., Windass J. D., Affara N., Paul J. Control of transcription of the globin gene. J Cell Physiol. 1975 Apr;85(2 Pt 2 Suppl 1):449–458. doi: 10.1002/jcp.1040850411. [DOI] [PubMed] [Google Scholar]
  12. Harris S. E., Schwartz R. J., Tsai M. J., O'Malley B. W., Roy A. K. Effect of estrogen on gene expression in the chick oviduct. In vitro transcription of the ovalbumin gene in chromatin. J Biol Chem. 1976 Jan 25;251(2):524–529. [PubMed] [Google Scholar]
  13. 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]
  14. Konkel D. A., Ingram V. M. RNA aggregation during sulfhydryl-agarose chromatography of mercurated RNA. Nucleic Acids Res. 1977 Jun;4(6):1979–1988. doi: 10.1093/nar/4.6.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lin R. I., Schjeide O. A. Micro estimation of RNA by the cupric ion catalyzed orcinol reaction. Anal Biochem. 1969 Mar;27(3):473–483. doi: 10.1016/0003-2697(69)90061-x. [DOI] [PubMed] [Google Scholar]
  16. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Minkley E. G., Pribnow D. Transcription of the early region of bacteriophage T7: selective initiation with dinucleotides. J Mol Biol. 1973 Jun 25;77(2):255–277. doi: 10.1016/0022-2836(73)90335-5. [DOI] [PubMed] [Google Scholar]
  18. Nguyen-Huu M. C., Sippel A. A., Hynes N. E., Groner B., Schütz G. Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B. Proc Natl Acad Sci U S A. 1978 Feb;75(2):686–690. doi: 10.1073/pnas.75.2.686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Wilson G. N., Steggles A. W., Kantor J. A., Nienhuis A. W., Anderson W. F. Cell-free transcription of mammalian chromatin. Quantitative measurement of newly synthesized globin messenger RNA sequences. J Biol Chem. 1975 Nov 25;250(22):8604–8613. [PubMed] [Google Scholar]
  21. 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]
  22. de Jiménez E. S., González J. L., Domínguez J. L., Saloma E. S. Characterization of DNA from differentiated cells. Analysis of the chicken genomic complexity. Eur J Biochem. 1974 Jun 1;45(1):25–29. doi: 10.1111/j.1432-1033.1974.tb03525.x. [DOI] [PubMed] [Google Scholar]

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