Identification of differentially methylated sequence and gene between white blood cells and sperm

Abstract

DNA methylation of cytosine within 5'CpG islands initiated in the germ line effects control of gene expression required for X-chromosome inactivation, genomics imprinting, and cell differentiation for normal embryonic development in mammals. The aberrant methylation occurs in the process of aging and carcinogenesis. Of the 30,000 genes contained within the mammalian genome, 2,000 are estimated to be regulated by DNA methylation, although only 40 genes have been clearly identified yet. The purpose of this study was to identify methylated DNA sequences by exhibiting different patterns upon CpG island of comparison between white blood cells and sperm. Employing a cross-sectional analytical study, we applied methylation-sensitive representational difference analysis (ms-RDA) to identify differentially methylated DNA sequences between white blood cells and sperm. The RDA product were inserted into a vector and cloned in E.coli. The DNA clones, obtained byamplified using the M13 primer, were tested as to this authenticity by Southern blot and subsequent hybridization to DNA extracted from white blood cells and sperm after treatment with methylation-sensitive restriction endonuclease, HpaII and its isozyme, MspI. We selected those hybridization products based on the autoradiogram hinted at differential methylation to direct sequencing and compare the data with the GenBank database. From 105 clones we found 6 clones were hypermethylated in blood compared with sperm. By DNA sequencing, we found that 3 clones had a GC content>50% and satisfied the minimal criteria for CpG islands (200 bp, GC content>50%, CpG/GpC>0.5). By analyzing the Blast program, there were 3 known human gene sequences were identical to these clones. There were cDNA sequences on 19q13.2 intergenic sequences of ribosomal DNA and 3' to exon 1 of Niemann-Pick C1 protein (NPCl) gene. However, no homology was found from the sixth.