c-Myc dependent genomic instability of the ribonucleotide reductase R2 gene

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Kuschak, Theodore I.
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c-Myc deregulation has been shown to generate locus-specific chromosomal and extrachromosomal gene amplification, as well as karyotypic instability. My studies focused on c-Myc-dependent amplification of a new c-Myc amplification target, the 'ribonucleotide reductase R2' gene. Using a mouse Pre-B cell line we showed chromosomal and extrachromosomal amplification and rearrangement of the 'R2' gene locus within 72 hours of transient and inducible c-Myc deregulation. We further showed that the initiation of c-Myc-dependent R2 instability occurs as early as 24 hours of transient c-Myc deregulation. Previous studies have demonstrated gene amplification using cell cycle inhibiting drugs, resulting in locus-specific gene amplification within 5-22 replication cycles. My study is novel in that it uses an inducible system to study initiation of c-Myc-dep ndent 'R2' amplification. The results of this work suggest that the c-Myc-dependent initiation of amplification of 'R2' is replication-driven. This does not exclude subsequent gene amplification through other mechanisms. To study c-Myc-dependent extrachromosomal gene amplification, we developed two methods for the analysis of extrachromosomal DNA amplicons. These methods include fluorescent 'in situ' hybridization to characterize the genes found in the total population of purified extrachromosomal DNA and the specific isolation of histone-bound extrachromosomal DNA, which is associated with active genes.