An apparent case of nonsymmetrical and sustained strand-specific hemimethylation in the Dc8 gene of carrot.

The Dc8 gene of carrot (Daucus carota L.) shows differential expression during embryo development. Changes in methylation patterns of a segment of about 500 bp (from base +120 to base -446) of Dc8 allele 6 were investigated by treating genomic DNA, extracted from embryogenic callus at different stages of development, with sodium bisulfite to modify nonmethylated cytosines. Following asymmetric (strand-specific) amplification, base sequences for samples from each developmental stage were determined for each strand directly from the PCR products or from cloned PCR products. Different methylation patterns were detected in the two strands. The 5' to 3' sense (coding) strand was almost completely nonmethylated, whereas almost all the cytosines in the 3' to 5' (template) strand were methylated. By 71 days after transfer to embryo-inducing medium, few methylcytosines remained; those that were present were generally near the TATA box or in a region beyond -300. The cytosines that were methylated were not limited to CG or CNG sequences. The difference in the extent of methylation between the two complementary strands implies either that there is a mechanism for strand-specific methylation, or that complementary sequences can differ greatly in sensitivity to bisulfite treatment or PCR amplification.