Structural analysis of a carcinogen-induced genomic rearrangement event

Abstract

We have explored the mechanism of genomic rearrangement in a hamster fibroblast cell culture system in which rearrangements are induced 5' to the endogenous thymidine kinase gene by chemical carcinogen treatment. The wild-type region around one rearrangement breakpoint was cloned and sequenced. With this sequence information, the carcinogen-induced rearrangement was cloned from the corresponding rearranged cell line by the inverse polymerase chain reaction. After the breakpoint fragment was sequenced, the wild-type rearrangement partner (RP15) was isolated by a second inverse polymerase chain reaction of unrearranged DNA. Comparison of the sequence of the rearrangement breakpoint with the wild-type RP15 and 5' thymidine kinase gene regions revealed short repeats directly at the breakpoint, as well as nearby A + T-rich regions in each rearrangement partner. Pulsed-field electrophoresis analysis demonstrated that this rearrangement is an interstitial deletion of 35 kilobases. Southern blot analysis of the RP15 region in unrearranged parental cells showed a demethylated CpG island and a complex of DNase I-hypersensitive sites adjacent to the breakpoint in the region deleted by the rearrangement. Therefore, these studies reveal interesting sequence and chromatin features near the rearrangement breakpoints and suggest a role for nuclear organization in the mechanism of carcinogen-induced genomic rearrangement.