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Purification of Plant Ribosomal DNA

Use of Buoyant Density Centrifugation in Cesium Chloride-Actinomycin D Gradients and DNA-RNA Hybridization
  • Donald Grierson
Protocol
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Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The general features of isopycnic centrifugation of DNA and the separation of main-band from satellite DNAs in solutions of cesium chloride are discussed in the preceeding chapter. This chapter describes a modification of the procedure that enhances the separation of specific DNA sequences from main-band DNA. The method is based on the fact that actinomycin D binds preferentially to DNA molecules with a high-GC content. This lowers their density relative to main-band and other satellite DNAs so that they can be resolved separately. Cesium chloride-actinomycin D gradients have been used to isolate sea urchin histone genes (1) and plant ribosomal DNA (2), and to separate genes coding for plant 25S and 18S from 5S RNA genes (3) and as a prelude to the cloning of ribosomal DNA (4).

Keywords

Satellite DNAs Nitrocellulose Filter Gradient Fraction Cesium Chloride Isopycnic Centrifugation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Birnstiel, M., Telford, J., Weinberg, E., and Stafford, D. (1974) Isolation and some properties of the genes coding for histone proteins. Proc. Natl. Acad. Sci. USA 71, 2900–2904.PubMedCrossRefGoogle Scholar
  2. 2.
    Hemleben, J., Grierson, D., and Dertmann, H. (1977) The use of equilibrium centrifugation in actinomycin-cesium chloride for the purification of ribosomal DNA. Plant Sci. Lett. 9, 129–135.CrossRefGoogle Scholar
  3. 3.
    Hemleben, V. and Grierson, D. (1978) Evidence that in higher plants the 25S and 185 rRNA genes are not interspersed with genes for 5S rRNA. Chromosoma (Berlin) 65, 353–358.CrossRefGoogle Scholar
  4. 4.
    Friedrich, J., Hemleben, V., Meagher, R.G., and Key, J.L. (1979) Purification and restriction endonuclease mapping of soybean 18S and 25S ribosomal RNA genes. Planta 146, 467–473.CrossRefGoogle Scholar
  5. 5.
    Grierson, D. (1982) RNA Processing and Other Posttranscriptional Events, in: Nucleic Acids and Proteins in Plants, Vol. II (Parthier, B. and Boulter, D., eds.) Springer-Verlag, Berlin-Heidelberg.Google Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • Donald Grierson
    • 1
  1. 1.Department of Physiology and Environmental SciencesUniversity of NotthinghamLoughboroughUK

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