| Research Abstract |
N-myc gene is frequently amplified in human Neuroblastoma(Nb) cells and correlation between N-myc gene amplification and advanced Nb tumor with poor prognosis is well documented. We have been working on the mechanism of N-myc gene amplification in Nb tumors and cell lines. We analyzed 7 cell lines (IMR-32, GOTO, TGW, Ngai, NB-1, YT-nu, SKNSH) and 86 tumor samples. Amplification of N-myc gene was observed in 5 cell line (IMR-32, GOTO, TGW, Nagai, YT-nu) and 9 tumor samples (StageIII2, StageIV_A 7) and DNA rearrangement in the vicinity of N-myc gene was also observed in 5 cell lines (GOTO, TGW, Ngai, YT-nu, NB-1). We isolated the rearranged DNA from TGW cells and determined the nucleotide sequences of the rearranged region. The rearrangement occurred between the end of exon 3 of N-myc gene and Alu sequence. The rearranged point in the Alu sequence corresponds to a hot point for DNA rearrangements including Alu-Alu homologous recombination. We also characterized N-myc gene amplification in three cell lines(IMR-32, TGW, GOTO). Rearrangements in long-range regions surrounding amplified N-myc genes were examined by pulsed-field gel electrophoresis. Since rarecutting enzymes complete-ly digested DNA at the middle of the N-myc gene, we were able to construct a physical map upstream and downstream of the germline N-myc gene, and to obtain information on restriction sites surrounding amplified N-myc genes. This method enables us to envisage the organization of amplified units over a long range. Digestion patterns differed considerably among the germline and the three cell lines, but were simple in each case.
We estimate that the minimal distance between neighboring N-myc genes is at least several hundred kilobases.
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