CPG ISLAND MAPPING OF A MOUSE DOUBLE-MINUTE CHROMOSOME

The development of double-minute chromosomes (DMs) and subsequent gene amplification are important genomic alterations resulting in increase d oncogene expression in a variety of tumors. The molecular mechanisms mediating the development of these acentric extrachromosomal elements have not been completely defined. To elucidate the mechanisms involve d in DM formation, we have developed strategies to map amplified circu lar DM DNA. In this study, we present a long-range restriction map of a 980-kb DM. A cell line cloned from mouse EMT-6 cells was developed b y stepwise selection for resistance to methotrexate. This cloned cell line contains multiple copies of the 980-kb DM carrying the dihydrofol ate reductase (DHFR) gene. A long-range restriction map was developed in which a hypomethylated CpG-rich region near the DHFR gene served as a landmark. This strategy was combined with plasmid-like analysis of ethidium bromide-stained pulsed-field gels and indicated that a single copy of the DHFR gene was located near a hypomethylated region contai ning SstII and NotI sites. At least 490 kb of this DM appears to be co mposed of unrearranged chromosomal DNA.