title: Analysis of rRNA Gene Methylation in Arabidopsis thaliana by CHEF-Conventional 2D Gel Electrophoresis.
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.
authors: Department of Biology, Indiana University, 915 E. Third Street, Bloomington, IN, 47405, USA. gmohanna@indiana.edu.|Department of Molecular and Cellular Biochemistry, Indiana University, 915 E. Third Street, Bloomington, IN, 47405, USA. gmohanna@indiana.edu.
authors: Gireesha
authors: G
authors: Mohannath
authors: Department of Biology, Indiana University, 915 E. Third Street, Bloomington, IN, 47405, USA.|Department of Molecular and Cellular Biochemistry, Indiana University, 915 E. Third Street, Bloomington, IN, 47405, USA.
authors: Craig S
authors: CS
authors: Pikaard
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D015139:Blotting, Southern; D019175:DNA Methylation; D004587:Electrophoresis, Agar Gel; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D020459:Genes, rRNA; D009697:Nucleolus Organizer Region / Q000235:genetics; D011523:Protoplasts; D012335:RNA, Ribosomal / Q000235:genetics
chemical_list: D012335:RNA, Ribosomal; C088179:RNA, ribosomal, 45S
keywords: 45S rRNA gene; Arabidopsis; Contour-clamped homogenous electric field (CHEF); DNA methylation; Nucleolus organizer region (NOR); Protoplast; Pulsed-field gel electrophoresis (PFGE); Southern blotting; Two-dimensional gel analysis; rDNA