ESTABLISHMENT OF METHYLATION-SENSITIVE-REPRESENTATIONAL DIFFERENCE ANALYSIS AND ISOLATION OF HYPO- AND HYPERMETHYLATED GENOMIC FRAGMENTS INMOUSE-LIVER TUMORS

Methylation of CpG sites in the genome, which is generally conserved d uring cell replication, is considered to play important roles in cell differentiation and carcinogenesis, However, investigations on changes in methylation status have been limited to known genes, To make a gen ome-wide search for differentially methylated genes, we developed a me thylation-sensitive-representational difference analysis (MS-RDA) meth od, The representation of the genome was prepared using the methylatio n-sensitive restriction enzyme HpaII, and the mixture ratio of tester and driver DNAs was optimized to detect differences in methylation sta tus of a single copy per diploid mammalian genome, By performing compa rative MS-RDA of one hepatocellular carcinoma and of background liver tissue of one mouse treated with a food carcinogen (2-amino-3,4-dimeth ylimidazo [4,5-f] quinoline), we were able to identify (i) extensive h ypomethylation of long interspersed nuclear element repetitive sequenc es in a number of hepatocellular carcinomas, (ii) reduction of the gen e dosage of their mitochondrial DNA, and (iii) a hypermethylated DNA f ragment of unknown origin, Furthermore, by adding the clones obtained in the first MS-RDA to the driver DNA [MS-RDA with elimination of exce ssive clones (MS-RDA-WEEC)], nine DNA fragments that could not be dete cted at the first MS-RDA were isolated as differentially methylated DN A fragments, MS-RDA, combined with MS-RDA-WEEC, is thus a promising ap proach to identify DNA fragments differentially methylated in two DNA sources.