Determination of the methylation status of CpG dinucleotides in theFugu locus containing the Fugu Surf-3/rpL7a, Surf-1,and Surf-6 genes by Southern blot analyses. The position ofMspI–HpaII (M/H), PstI (P), andHindIII (H3) restriction enzyme recognition sites are shown along the 9.4 kb of sequence (horizontal line) obtained for the locus containing the Fugu Surf-3/rpL7a, Surf-1, and Surf-6genes. The predicted positions of other MspI–HpaII restriction sites outside the region sequenced are shown above a broken line and are included to facilitate interpretation of the data. The relative positions and orientations of each gene from initiator methionine to termination codon (except Surf-1 whose 5′ end is not determined) are indicated by bold arrows. Probes derived from PstI, HindIII, orPstI–HindIII restriction fragments used in the Southern blot analyses are shown as shaded boxes below and are noted as spanning MspI–HpaII restriction sites. (A–D) A Southern blot analysis probed with the corresponding probe (A–D). For each blot, 0.4 ng of cosmid 186H17 DNA was digested with MspI (lane 1), 3 μg ofFugu genomic DNA was digested with MspI (lane2), and HpaII (lane 3) and run on either a 2% agarose gel (A) or a 1% agarose gel (B,C,D).MspI and HpaII restriction digests of cosmid 186H17 DNA give an identical pattern of restriction fragments indicating that the cloned cosmid DNA contains no MspI–HpaII restriction sites containing methylated CpG dinucleotides (data not shown). Probable methylated CpG dinucleotides withinMspI–HpaII restriction sites as determined by this analysis are indicated by an asterisk (*). (§) At least one of the three MspI–HpaII restriction sites at theSurf-1/Surf-6 promoters is predicted not to be methylated.
