Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1977 Jun;4(6):1979–1988. doi: 10.1093/nar/4.6.1979

RNA aggregation during sulfhydryl-agarose chromatography of mercurated RNA.

D A Konkel, V M Ingram
PMCID: PMC342535  PMID: 896479

Abstract

Isolation of newly synthesized mercurated RNA transcripts by chromatography on sulfhydryl-agarose has recently been used to reduce contamination by endogenous RNA derived from the chromatin template. We show that substantial RNA aggregation occurs during standard isolation procedures, causing significant retention of endogenous (unmercurated) RNA on sulfhydryl-agarose. We describe methods to reduce substantially this problem and discuss the implications of our findings for interpretation of previous hybridization and transcription experiments.

Full text

PDF

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. Burgess R. R. A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6160–6167. [PubMed] [Google Scholar]
  6. Chan L. N., Wiedmann M., Ingram V. M. Regulation of specific gene expression during embryonic development: Synthesis of globin messenger RNA during red cell formation in chick embryos. Dev Biol. 1974 Sep;40(1):174–185. doi: 10.1016/0012-1606(74)90117-1. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Comings D. E., Harris D. C. Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei. J Cell Biol. 1976 Aug;70(2 Pt 1):440–452. doi: 10.1083/jcb.70.2.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  11. 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]
  12. Dale R. M., Ward D. C. Mercurated polynucleotides: new probes for hybridization and selective polymer fractionation. Biochemistry. 1975 Jun 3;14(11):2458–2469. doi: 10.1021/bi00682a028. [DOI] [PubMed] [Google Scholar]
  13. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  14. Gilmour R. S., MacGillivray A. J. The role of non histone proteins in the regulation of globin gene activity. Symp Soc Dev Biol. 1976;(34):15–27. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Groudine M., Holtzer H., Scherrer K., Therwath A. Lineage-dependent transcription of globin genes. Cell. 1974 Nov;3(3):243–247. doi: 10.1016/0092-8674(74)90138-x. [DOI] [PubMed] [Google Scholar]
  18. Moss B. A., Joyce W. G., Ingram V. M. Histones in chick embryonic erythropoiesis. J Biol Chem. 1973 Feb 10;248(3):1025–1031. [PubMed] [Google Scholar]
  19. Nicolini C., Baserga R. Conformational changes in chromatin from density inhibited WI-38 fibroblasts stimulated to proliferate. Ric Clin Lab. 1975 Jul-Sep;5(3):219–233. doi: 10.1007/BF02908286. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Swan D., Aviv H., Leder P. Purification and properties of biologically active messenger RNA for a myeloma light chain. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1967–1971. doi: 10.1073/pnas.69.7.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. de Pomerai D. I., Chesterton C. J., Butterworth P. H. Preparation of chromatin. Variation in the template properties of chromatin dependent on the method of perparation. Eur J Biochem. 1974 Aug 1;46(3):461–471. doi: 10.1111/j.1432-1033.1974.tb03639.x. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES