Differential methylation of Pl-Bh appears to be restricted to cytosines in CG or CNG sequences
-- Michael G. Muszynski and Karen C. Cone

Pl-Bh is hypermethylated at specific MspI/HpaII sites 3' of the coding region compared to Pl (Figure 8, Cocciolone and Cone, Genetics 135:575-588, 1993). The MspI/HpaII restriction site is CCGG. MspI is sensitive to methylation at the internal cytosine, while HpaII is sensitive to methylation at either cytosine. Both cytosines are located within either CG or CNG sequences, which are the preferred targets for DNA methylation in plants. Methylation of cytosines outside of these target sequences has been reported for transgenes in petunia (P. Meyer et al., EMBO J. 13:2084-2088, 1994). This observation prompted us to assay for differential cytosine methylation outside of the canonical CG or CNG sequences in Pl and Pl-Bh. The restriction enzymes AluI (AGCT), Sau3AI (GATC) and Sau96I (GGNCC), which are all sensitive to cytosine methylation, were used. The restriction sites assayed are located 3' of the coding region and none have a cytosine in a CG or CNG context. Digestion with these enzymes was complete for both Pl and Pl-Bh . This indicates that there are no methylated cytosines at these sites in Pl and Pl-Bh. Therefore, Pl-Bh appears to only be differentially methylated within CG or CNG sequences. Methods exist for detecting every methylated cytosine of a target sequence (S. Clark et al., NAR 22:2990-2997, 1994). We are currently adapting these methods for use on maize DNA to detect and quantitate methylation at individual cytosines in Pl and Pl-Bh.
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