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 carcinogeninduced genomic rearrangement.Numerous sporadic rearrangements have been identified in mammalian cells, both as acquired changes in tumors and as constitutional changes in heritable diseases. Characterization of the sequences at and near the breakpoints has provided evidence of multiple mechanisms of genomic rearrangement. Postulated mechanisms include mistakes in V(D)J recombination (1), homologous recombination between repetitive sequences (2), and nonhomologous recombination between regions with only 1-to 7-nucleotide blocks of junctional homology (3). Sequences capable of forming non-B-DNA structures, such as A+T-rich regions (4), homopurine and homopyrimidine tracts (5), and stretches of alternating purines and pyrimidines (6), as well as nuclear matrix attachment sites (7), have been identified near several rearrangement breakpoints and are postulated to disrupt chromatin structure and enhance accessibility for recombination.Though sequence analysis of rearranged cell lines has yielded interesting clues, these systems are inherently limited. Since the rearrangement event occurred long before the cell line was derived, the nuclear organization of the parental cell type and the environmental conditions associated with the event are unknown. A better system for analyzing the mechanism of genomic rearrangement would be an inducible system in which a uniform cell population is exposed to an environmental perturbation resulting in a detectable rearrangement frequency at a locus ofinterest. A system in which the quiescent endogenous thymidine kinase (TK) gene of RJK92 Chinese hamster cells can be activated and rearranged following chemic...

Fusion genes resulting from alternative chromosomal translocations are overexpressed by gene-specific mechanisms in alveolar rhabdomyosarcoma

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“…45 However, successful amplifications were limited to template sizes Ͻ1 kb. 45 We believe that panhandle PCR is a technical advance towards understanding the molecular pathogenesis of leukemias with MLL gene translocations. Although panhandle PCR contains several steps including multiple glass-bead extractions, the initial success demonstrates that panhandle PCR is a straightforward method to clone MLL genomic breakpoints.…”

“…Inverse PCR has also been used to amplify a carcinogen-induced thymidine kinase gene rearrangement. 45 The approach included restriction enzyme cleavage on each side of the rearrangement, circularization of the restriction fragment with T4 DNA ligase, and PCR amplification with outward primers both derived from the known side of the rearrangement. 45 However, successful amplifications were limited to template sizes Ͻ1 kb.…”

“…45 The approach included restriction enzyme cleavage on each side of the rearrangement, circularization of the restriction fragment with T4 DNA ligase, and PCR amplification with outward primers both derived from the known side of the rearrangement. 45 However, successful amplifications were limited to template sizes Ͻ1 kb. 45 We believe that panhandle PCR is a technical advance towards understanding the molecular pathogenesis of leukemias with MLL gene translocations.…”