5-Lipoxygenase DNA Methylation and mRNA Content in the Brain and Heart of Young and Old Mice

Figure 1

The sequence of the mouse 5-LOX promoter-5^′ UTR and the first exon-intron region subjected to the methylation-sensitive endonuclease assay with the four endonucleases—AciI, BstUI, HinP1I, and HpaII. The yellow highlight indicates the first exon including the start codon (ATG, in red). The four endonuclease-targeted methylation-sensitive endonuclease recognition sites are highlighted by four different colors. The selected DNA sequence included 2 AciI-sensitive sites (upstream of the start codon), 6 BstUI-sensitive sites (2 upstream and 4 downstream of the start codon), 8 HinP1I-sensitive sites (2 upstream and 6 downstream of the start codon), and 3 HpaII-sensitive sites (2 upstream and 1 downstream of the start codon). The primer regions used for quantitative PCR amplification are underlined; primers were used as follows. Promoter - 5^′ UTR: forward = 5^′-aga gaa gga tgc gtt gga aggt-3^′ and reverse = 5^′-gac tcc ggg caa gtg agt gct-3^′; exon-intron: forward = 5^′-agt cat gcc ctc cta cac ggt ca-3^′ and reverse = 5^′-agt cat gcc ctc cta cac ggt ca-3^′. For the input control, we used the following primers: forward = 5^′-tga tgt ggc tgg cct ctt atg tga-3^′, reverse = 5^′-act ggg act gag tgc agg aaa tgt-3^′.