Related Experiment Videos
Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma
S S Schneider1, J L Hiemstra, B A Zehnbauer
1Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
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.
Insights
Gene amplification in neuroblastoma involves a circular 1.2-Mb N-myc amplified domain. This structure, with conserved gene mapping, suggests an episomal amplification mechanism in tumor cells.
Area of Science:
- Molecular Biology
- Cancer Genetics
- Genomics
Background:
- Oncogene amplification is common in human cancers, but its mechanisms and DNA structures remain unclear.
- Neuroblastoma frequently exhibits N-myc oncogene amplification, driving tumor progression.
Purpose of the Study:
- To investigate the structure of the N-myc amplified domain in a neuroblastoma cell line (SMS-KAN).
- To compare the physical map of the amplified domain with the normal N-myc locus.
- To elucidate the mechanism of oncogene amplification in neuroblastoma.
Main Methods:
- Cloning of neuroblastoma DNA into yeast artificial chromosomes (YACs).
- Screening YAC libraries using amplified DNA probes.
- Comparative restriction mapping of YACs representing the amplified domain and the normal N-myc locus.
Main Results:
- The amplified N-myc domain in SMS-KAN cells is approximately 1.2 Mb and exists as a circular molecule.
- The amplified DNA exhibits a head-to-tail configuration.
- The physical map of the normal N-myc locus is generally conserved within the amplicon.
Conclusions:
- The study provides a physical map of the amplified oncogene domain in a neuroblastoma cell line.
- The circular, head-to-tail organization and extrachromosomal location support the episome formation-plus-segregation model for gene amplification.
- Findings offer insights into the molecular mechanisms driving oncogene amplification in neuroblastoma.