Selection and sequencing of interchromosomal rearrangements from gamma-irradiated normal human fibroblasts

Abstract

Purpose : To determine the sequences that flank sites of interchromosomal DNA rearrangements and to determine the relative frequency of inter- and intrachromosomal rearrangements induced by 30Gy gamma -irradiation in a region 5 from exon I of the c-myc gene in normal human fibroblasts (IMR-90). Materials and methods : A modification of an inverse polymerase chain reaction (PCR) procedure, developed previously to detect rearrangements, was used. Inverse PCR products were re-amplified using primers designed to determine whether the product was a result of an inter- or intrachromosomal rearrangement. Possible interchromosomal rearrangements were then sequenced. Results and conclusions : Four of 12 different products analyzed were potentially derived from interchromosomal rearrangements, while the remainder derived from intrachromosomal rearrangements. For three of the potential interchromosomal rearrangements, the sequence recombining with c-myc was unidentified, while in the other case the sequence was homologous to an L1 element. The frequencies of inter- and intrachromosomal rearrangements induced by 30Gy gamma -irradiation in a 2kbp region flanking the c-myc gene of IMR-90 cells were calculated to be at least 1.6 10 4 and 3.3 10 4 respectively. No clear association between sequence context and sites of radiation-induced rearrangement was found; however, two of the four sequenced rearrangements involved breakpoints in the 5-flanking region of c-myc that occurred immediately after the sequence AAAGG.