Figure 3.

Restriction endonuclease analysis to determine methylation status of MAGGY sequences in R0–R8 regenerants (A). Genomic DNA extracted from the regenerants was digested with isoschizomers HapII (H) and MspI (M). HapII is sensitive to cytosine methylation in the recognition sequence CCGG but MspI is not. HapII and MspI digests were separated in an agarose gel and hybridized to a 5.4 kb XhoI fragment of MAGGY (Fig. 1A). Complete digestion with the enzymes was checked by reprobing with pEBA18 containing a LINE-like retrotransposon, MGR583 (24) (data not shown). (B) For accurate comparison of the degree of methylation in the MAGGY sequence, the concentrations of genomic DNA of R1, R2, R7 and R8 were adjusted according to the copy numbers of MAGGY they possessed and subjected to analysis as in (A).