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Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis

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Book cover The Nucleolus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1455))

Abstract

Contour-clamped homogenous electric field (CHEF) gel electrophoresis, a variant of Pulsed-field gel electrophoresis (PFGE), is a powerful technique for resolving large fragments of DNA (10 kb–9 Mb). CHEF has many applications including the physical mapping of chromosomes, artificial chromosomes, and sub-chromosomal DNA fragments, etc. Here, we describe the use of CHEF and two-dimensional gel electrophoresis to analyze rRNA gene methylation patterns within the two ~4 million base pair nucleolus organizer regions (NORs) of Arabidopsis thaliana. The method involves CHEF gel electrophoresis of agarose-embedded DNA following restriction endonuclease digestion to cut the NORs into large but resolvable segments, followed by digestion with methylation-sensitive restriction endonucleases and conventional (or CHEF) gel electrophoresis, in a second dimension. Resulting products are then detected by Southern blotting or PCR analyses capable of discriminating rRNA gene subtypes.

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Authors and Affiliations

  1. Department of Biology, Indiana University, 915 E. Third Street, Bloomington, IN, 47405, USA

    Gireesha Mohannath & Craig S. Pikaard

  2. Department of Molecular and Cellular Biochemistry, Indiana University, 915 E. Third Street, Bloomington, IN, 47405, USA

    Gireesha Mohannath & Craig S. Pikaard

Authors
  1. Gireesha Mohannath
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  2. Craig S. Pikaard
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Correspondence to Gireesha Mohannath .

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Editors and Affiliations

  1. Department of Biochemistry III, University of Regensburg, Regensburg, Germany

    Attila Németh

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Mohannath, G., Pikaard, C.S. (2016). Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis. In: Németh, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_14

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  • DOI: https://doi.org/10.1007/978-1-4939-3792-9_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3790-5

  • Online ISBN: 978-1-4939-3792-9

  • eBook Packages: Springer Protocols

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