The RLGS spots were cloned using the restriction trapper-based RLOS spot

Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect, and subsequently clone, genomic regions with alter ations in DNA methylation associated with tumorigenesis. Use of a meth ylation-sensitive enzyme for the landmark cleavage allows analysis of changes in methylation patterns. In this study, we used RLGS-M to analyze SV4OT antigen-induced mouse liver tumors derived from inter specificF1 hybrids betweenMus spretus (S) and CS7BLI6(B6).Because 575 Sand B6-specific RLGS loci/spots have been mapped, tumor-related alterations in the RLGS profile could be immediately localized to specific chromosomal regions. We previously found that the loss of contiguous led/spots could be attributed primarily to DNA loss, whereas loss of solitary loci/spots could be attributed primarily to DNA methylation In this study, we examined 30 mouse liver tumor samples for loss of the 507 mapped loci/spots. Fourteen solitary IeeE/spots found to be absent or reduced in more than 75% oftumor samples were cloned and subjected to DNA sequence analyses Two loci were identified as a4 integrin and p16/CDKN2, genes reported to be involved in tumorigenesis. Thus, RLGS-M can detect alterations in the methylation status of known tumor suppressor genes and provide a method for detecting and subsequently cloning novel genomic regions that undergo alterations in methylation during tumongenesis.