Fig. 1 High levels of DNA methylation in gametes and early embryonic stages of zebrafish. a, A Southern blot of DNA samples (100 ng) from sperm, adult tail and embryonic stages (2.2 h–5 d) hybridized with the DANA repeat probe. After digestion with EcoRI (R), EcoRI/HpaII (H) or EcoRI/MspI (M), digests were assayed for completeness by co-incubating an aliquot (1/10) of the mixture with plasmid DNA (100 ng). Southern blots were prepared and hybridized using standard protocols. The DANA repeat probe was prepared by amplification of genomic DNA using primer sequences Dana-1, 5–GGCGACRCAGTGGCGCAGTRGG–3, and Dana-2, 5–TTTTCTTTTTGGCTTAGTCCC–3 (ref. 6). b, Map-plots of CpG distribution (top line) and position of sites tested for methylation at fgf-3, whn and rag-1. Vertical lines indicate the position of CpG dinucleotides. Methylated sites in adult DNA, as determined from Southern blots, are indicated by filled circles; partially methylated sites by half-filled circles. The regions tested by genomic sequencing are bracketed. c, Bisulfite sequence analysis of all CpGs in above segments of fgf-3, whn and rag-1. The CpGs in each DNA sequence are represented by filled circles (methylated) and open circles (non-methylated). Methylation of rag1 in the egg sample was not analysed. The number of clones with each methylation pattern is shown on the left. Genomic sequence by bisulfite modification was obtained using standard procedures5, 11. The amplified DNA was cloned into the pGEM–T Easy vector (Promega) and sequenced using a PE-Applied Biosystems 373A sequencer. a b c

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