Figure 3
Molecular structure and differentially methylated region (DMR), BSP amplified nucleotide sequences and DNA methylation patterns of IGF2 DMR1.
(A) Molecular structure of the bovine IGF2 gene. Intron 3 and exon 10 contains two differentially methylated regions (DMRs). A 2000-bp fragment centred around intron 3–3106 (intron 3–3072 mutation on pig IGF2) was considered to methylation analysis. (B) Schematic representation of the proximal promoter region (IGF2 intron 3 −2106 to −4105 base pairs) of the bovine IGF2 gene (modified output of MethPrimer program) to predict regions of high GC content. Dashed lines indicate the GC percentage as represented on the y-axis and the x-axis denotes the bp position on the IGF2 gene intron 3 −2106 to −4105 base pairs untranslated region. Arrows indicate the intron 3–3106 mutation. Coordinates are given in relation to the intron 3–3106 mutation site (shown as +1); vertical lines indicate relative positions of CpG dinucleotides; solid lines depict location of the PCR primers. The 793 bp CpG islands are evident in the intron 3 of the gene, with GC content of 62.50%. (C) Nucleotide sequences for a 366 bp IGF2 DMR (total 54 CpG site) fragment (upper strands) and its bisulfite-converted version (lower strands). Primer sequences are underlined. (D) DNA methylation patterns of muscle tissue in the fetal bovine (FB: 23.1%) group and adult bovine (AB: 33.2%) group analyzed by BSP. Each line represents one individual bacterial clone and each circle one single CpG dinucleotide. Open circles show unmethylated CpG's and black circles methylated CpG's. The gray box show the 28th CpG site of the intron 3–3106 mutation site by BSP analyses.
