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.

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.

Figure 1

CHEF gel orientations: Wide gel (on the left side) allows more space for samples with less resolving distance where in long gel (on the right side) provides less sample space with longer resolving distance.

Figure 2

CHEF gel electrophoresis of HindIII-digested agarose-embedded DNA of Arabidopsis thaliana ecotype Col-0 (first dimension). On the left side is an ethidium bromide-stained gel image. On the right side is the image obtained after southern blotting and probing with rRNA gene radioactive probe. M1=GeneRuler 1kb Plus DNA ladder; M2=Yeast Chromosome PFG marker (NEB: N0345S). The topmost band in the M2 lane corresponds to yeast (Saccharomyces cerevisiae) chromosome 12, which contains ribosomal genes [28] and cross-reacts with the probe. Also, some fragments in M1 cross-react with the probe.

Figure 3

Setting up a CHEF gel slice for in-gel restriction digestion.

Figure 4

Casting a new agarose gel with a CHEF gel slice at the top, for the second dimension gel electrophoresis

Figure 5

Two-dimensional gel electrophoresis of agarose-embedded DNA of Arabidopsis thaliana ecotype Col-0. CHEF gel electrophoresis of HindIII-digested products in the first dimension and conventional gel electrophoresis of non-digested (image at the top) or HpaII (methylation-sensitive endonuclease)-digested products (image at the bottom) in the second dimension. The DNA fragments marked with dotted circles are hypomethylated compared to the DNA fragments marked with undotted circles. M= HpaII-digested products of 4 Arabidopsis rRNA gene-containing BAC clones [27].