Detection and sequencing of ionizing radiation-induced DNA rearrangements using the inverse polymerase chain reaction.
OBJECTIVE
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.


METHODS
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.
Forrester
H B
HB
Sir Donald and Lady Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Victoria, Australia.
Radford
I R
IR
D001483:Base Sequence; D019457:Chromosome Breakage / Q000235:genetics; D002875:Chromosomes / Q000528:radiation effects; D004247:DNA / Q000528:radiation effects*; D004252:DNA Mutational Analysis / Q000379:methods*; D004260:DNA Repair / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000235:genetics; D005347:Fibroblasts; D005720:Gamma Rays; D016259:Genes, myc / Q000528:radiation effects; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011839:Radiation, Ionizing; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors
D004247:DNA; D004264:DNA Topoisomerases, Type I