H B Forrester1, I R Radford. 1. Sir Donald and Lady Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Abstract
PURPOSE: To develop a procedure, using the inverse polymerase chain reaction, to detect and sequence ionizing radiation-induced DNA rearrangements without prior phenotypic selection of mutant cells. METHOD: Normal human fibroblast cells (IMR-90) were given 30Gy of gamma-irradiation and then incubated at 37 degrees C for 23h to allow DNA repair. Rearrangements of the sequence 5' to the c-myc gene were examined by amplifying the region using inverse PCR followed by DNA sequencing. RESULTS: Approximately fivefold more PCR products were amplified from the DNA of cells given 30 Gy of gamma-irradiation and allowed 23 h for repair than were obtained from cells that were either unirradiated or were irradiated and then lysed immediately. PCR products from seven putative radiation-induced DNA rearrangements were sequenced. Of these products, one contained an unidentified sequence (a possible inter-chromosomal rearrangement) whilst the other products appeared to derive from episomes or duplication events (possible intra-chromosomal rearrangements). The sequencing data suggested that the sites of DNA rearrangement breakpoints were non-randomly distributed and possibly associated with topoisomerase I consensus cleavage sequences. There was a significant level of direct homology between the sequences flanking the breakpoints. CONCLUSIONS: The procedure developed was able to detect both inter- and intra-chromosomal rearrangements.
PURPOSE: To develop a procedure, using the inverse polymerase chain reaction, to detect and sequence ionizing radiation-induced DNA rearrangements without prior phenotypic selection of mutant cells. METHOD: Normal human fibroblast cells (IMR-90) were given 30Gy of gamma-irradiation and then incubated at 37 degrees C for 23h to allow DNA repair. Rearrangements of the sequence 5' to the c-myc gene were examined by amplifying the region using inverse PCR followed by DNA sequencing. RESULTS: Approximately fivefold more PCR products were amplified from the DNA of cells given 30 Gy of gamma-irradiation and allowed 23 h for repair than were obtained from cells that were either unirradiated or were irradiated and then lysed immediately. PCR products from seven putative radiation-induced DNA rearrangements were sequenced. Of these products, one contained an unidentified sequence (a possible inter-chromosomal rearrangement) whilst the other products appeared to derive from episomes or duplication events (possible intra-chromosomal rearrangements). The sequencing data suggested that the sites of DNA rearrangement breakpoints were non-randomly distributed and possibly associated with topoisomerase I consensus cleavage sequences. There was a significant level of direct homology between the sequences flanking the breakpoints. CONCLUSIONS: The procedure developed was able to detect both inter- and intra-chromosomal rearrangements.
Authors: Raymund L Yong; Chunzhang Yang; Jie Lu; Huaien Wang; Cody D Schlaff; Anita Tandle; Christian A Graves; Abdel G Elkahloun; Xiaoyuan Chen; Zhengping Zhuang; Russell R Lonser Journal: Nat Commun Date: 2014-12-17 Impact factor: 14.919