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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Cox EC, Vocke CD, Walter S, Gregg KY, Bain ES (1990) Electrophoretic karyotype for Dictyostelium discoideum. Proc Natl Acad Sci U S A 87(21):8247–8251
Schwartz DC, Cantor CR (1984) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37(1):67–75
Chu G, Vollrath D, Davis RW (1986) Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234(4783):1582–1585
O’Brien FG, Udo EE, Grubb WB (2006) Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus. Nat Protoc 1(6):3028–3033. doi:10.1038/nprot.2006.382
Copenhaver GP, Doelling JH, Gens S, Pikaard CS (1995) Use of RFLPs larger than 100 kbp to map the position and internal organization of the nucleolus organizer region on chromosome 2 in Arabidopsis thaliana. Plant J 7(2):273–286
Ryan AJ, Squires S, Strutt HL, Johnson RT (1991) Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double strand breaks in replicating DNA. Nucleic Acids Res 19(12):3295–3300
Bautsch W (1992) Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE). In: Burmeister M, Ulanovsky L (eds) Methods in molecular biology: pulsed-field gel electrophoresis, vol 12. Humana Press Inc., Totowa, NJ, pp 185–201
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517
Southern EM (1992) Detection of specific sequences among DNA fragments separated by gel electrophoresis. 1975. Biotechnology 24:122–139
Warburton PE, Wevrick R, Mahtani MM, Willard HF (1992) Pulsed-field and two dimensional gel electrophoresis of long arrays of tandemly repeated DNA. In: Burmeister M, Ulanovsky L (eds) Methods in molecular biology: pulsed-field gel electrophoresis, vol 12. Humana Press Inc., Totowa, NJ, pp 299–317
Copenhaver GP, Pikaard CS (1996) Two-dimensional RFLP analyses reveal megabase-sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution. Plant J 9(2):273–282
Copenhaver GP, Pikaard CS (1996) RFLP and physical mapping with an rDNA-specific endonuclease reveals that nucleolus organizer regions of Arabidopsis thaliana adjoin the telomeres on chromosomes 2 and 4. Plant J 9(2):259–272
Goering RV (1993) Molecular epidemiology of nosocomial infection: analysis of chromosomal restriction fragment patterns by pulsed-field gel electrophoresis. Infect Control Hosp Epidemiol 14(10):595–600
Phillips RL, Kleese RA, Wang SS (1971) The nucleolus organizer region of maize (Zea mays L.): chromosomal site of DNA complementary to ribosomal RNA. Chromosoma 36(1):79–88. doi:10.1007/BF00326423
Raska I, Shaw PJ, Cmarko D (2006) Structure and function of the nucleolus in the spotlight. Curr Opin Cell Biol 18(3):325–334. doi:10.1016/j.ceb.2006.04.008
Long EO, Dawid IB (1980) Repeated genes in eukaryotes. Annu Rev Biochem 49:727–764. doi:10.1146/annurev.bi.49.070180.003455
Albini SM (1994) A karyotype of the Arabidopsis thaliana genome derived from synaptonemal complex analysis at prophase I of meiosis. Plant J 5(5):665–672. doi:10.1111/j.1365-313X.1994.00665.x
Earley KW, Pontvianne F, Wierzbicki AT, Blevins T, Tucker S, Costa-Nunes P, Pontes O, Pikaard CS (2010) Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation. Genes Dev 24(11):1119–1132. doi:10.1101/gad.1914110
Lawrence RJ, Pikaard CS (2004) Chromatin turn ons and turn offs of ribosomal RNA genes. Cell Cycle 3(7):880–883
Pontvianne F, Abou-Ellail M, Douet J, Comella P, Matia I, Chandrasekhara C, Debures A, Blevins T, Cooke R, Medina FJ, Tourmente S, Pikaard CS, Saez-Vasquez J (2010) Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana. PLoS Genet 6(11):e1001225. doi:10.1371/journal.pgen.1001225
Pontvianne F, Blevins T, Chandrasekhara C, Mozgova I, Hassel C, Pontes OM, Tucker S, Mokros P, Muchova V, Fajkus J, Pikaard CS (2013) Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes Dev 27(14):1545–1550. doi:10.1101/gad.221648.113
Doelling JH, Gaudino RJ, Pikaard CS (1993) Functional analysis of Arabidopsis thaliana rRNA gene and spacer promoters in vivo and by transient expression. Proc Natl Acad Sci U S A 90(16):7528–7532
Doelling JH, Pikaard CS (1993) Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures. Plant Cell Rep 12(5):241–244. doi:10.1007/bf00237127
Bio-Rad L (1992) CHEF DRIII pulsed field electrophoresis systems: instructions manual and applications guide. Bio-Rad Laboratories, Hercules, CA
Southern E (2006) Southern blotting. Nat Protoc 1(2):518–525. doi:10.1038/nprot.2006.73
Van Daelen R, Zabel P (1992) Pulsed-field gel electrophoresis. In: Beckman JS, Osborn TC (eds) Plant genomes. Kluwer Academic Publishers, Dordrecht, pp 71–100
Mozo T, Fischer S, Shizuya H, Altmann T (1998) Construction and characterization of the IGF Arabidopsis BAC library. Mol Gen Genet 258(5):562–570
Machin F, Torres-Rosell J, Jarmuz A, Aragon L (2005) Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase. J Cell Biol 168(2):209–219. doi:10.1083/jcb.200408087
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3792-9_14
Published:
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