ABSTRACT
Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.
Subject(s)
Gene Amplification , Genes, myc , Neuroblastoma/genetics , Base Sequence , Chromosomes, Fungal , Cloning, Molecular , DNA, Neoplasm , Genome, Human , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Restriction MappingABSTRACT
The Huntington's disease (HD) gene has been localized by recombination events to a region covering 2.2 megabases (Mb) DNA within chromosome 4p16.3. We have screened three yeast artificial chromosome (YAC) libraries in order to isolate and characterize 44 YAC clones mapping to this region. Approximately 50% of the YACs were chimaeric. Unstable YACs were identified across the whole region, but were particularly prevalent around the D4S183 and D4S43 loci. The YACs have been assembled into a contig extending from D4S126 to D4S98 covering roughly 2 Mb DNA, except for a gap of about 250 kilobases (kb). The establishment of a YAC contig which spans the region most likely to contain the HD mutation is an essential step in the isolation of the HD gene.
