This application focuses on utilizing Restriction Landmark Genomic Scanning (RLGS) to detect genetic and epigenetic alterations which contribute to multi-step tumorigenesis. RLGS can effectively detect gene amplification, gene loss, and is unique in its ability to detect alterations in DNA methylation. Approximately 2,000 loci distributed across the entire genome can be detected in a single analysis. The ability to """"""""spot clone"""""""" directly from the RLGS gel allows immediate access to the genomic regions of interest. Experiments are proposed to utilize two model mouse tumor systems, liver tumorigenesis in the Major Urinary Protein (MUP)/SV40 T antigen transgenic mice, and intestinal tumorigenesis in the Multiple Intestinal Neoplasia (Min) mouse which has a mutation in the murine homolog of the APC gene. The model mouse tumor systems allow a somewhat controlled tumorigenetic pathway in a defined genetic background which can be manipulated experimentally. Tumor related alterations can be rapidly localized to specific chromosomal loci. Experiments will concentrate on identifying and isolating spot clones and other genomic clones corresponding to genetic loci in which both copies are frequently deleted and/or hypermethylated during tumorigenesis. Genes within these regions will be identified to establish whether the genetic alteration (gene loss, methylation, or amplification) alters gene function during tumorigenesis. Initial studies will concentrate on regions identified by two spot clones, S238 which was found to correspond to p161NK4a and B330 which identifies a genomic region on chromosome 15 (S15Ncvs1) that undergoes strain and tumor specific alterations in methylation. These studies should provide a means for identifying novel genes involved in tumorigenesis and further our understanding of the role of DNA methylation in this process.
