Methylation of the <i>A^vy</i> Allele following Paternal Transmission

<div><p>The methylation status of each CpG dinucleotide was determined by sequencing PCR clones of bisulfite-converted DNA [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b004" target="_blank">4</a>]. Each line represents an individual clone, theoretically from one cell, and each circle an individual CpG. Open circles indicate an unmethylated CpG, and closed circles a methylated CpG. Each block of lines represents the clones derived from the sperm of one adult male mouse, one set of ten zygotes, ten two-cell embryos or one blastocyst. The percentage of methylation in each dataset is shown (calculated from the number of methylated CpGs divided by the total CpGs sequenced, multiplied by 100). The position of each circle is representative of the relative location along the length of the PCR product. Any clones with greater than 5% non-CpG methylation were excluded from the dataset, and these clones made up less than 5% of all clones sequenced. These clones tended to have very high levels of non-CpG methylation, an indication of incomplete bisulfite conversion. Zygotes, two-cell embryos, or blastocysts were collected from yellow or pseudoagouti <i>A^vy/a</i> sires mated to <i>a/a</i> dams, as indicated. Clones were only included in the zygote or blastocyst samples if they could be distinguished from others in the sample by CpG or low-level non-CpG methylation.</p><p>(A) The IAP LTR pseudoexon 1a junction shown in detail. The bisulfite sequencing primers are shown [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020049#pgen-0020049-b004" target="_blank">4</a>]. The PCR product contains 11 CpG dinucleotides, depicted as circles, all of which are in the LTR.</p><p>(B) Data obtained from the sperm of four pseudoagouti males, and 10 × 10 zygotes, 10 × 10 two-cell embryos or 10 × 1 blastocysts collected from pseudoagouti <i>A^vy/a</i> sires mated to <i>a/a</i> dams.</p><p>(C) Data obtained from the sperm of four yellow male and 9 × 1 blastocysts collected from yellow <i>A^vy/a</i> sires mated to <i>a/a</i> dams. These data indicate that the <i>A^vy</i> allele is subject to rapid demethylation immediately postfertilisation following paternal transmission.</p>