posted on 2014-12-15, 10:34authored byRachel Caroline Le Pla
Previously, in this laboratory, it has been demonstrated that the manifestation of PTEs was non-random in vivo, but random in vitro with respect to normal base content. This was suggestive of either non-random formation in vivo or sequence specific repair (Guichard et al., 2000). In the present study, the [32P]-postlabelling protocol was further developed to allow for the more straight forward determination of nucleosides 5' to the PTEs. The molecular basis for the non-random manifestation of PTEs in vivo was studied using the improved assay. To determine the influence of cellular processes upon the manifestation of PTEs in liver DNA take from mice 5 hours to 56 days following a single dose of N-nitrosodiethylamine was analysed. Whilst there was an abrupt decline in the level of PTEs observed, possible indicative of active repair, there was no appreciable change in the frequency of nucleosides located 5' to PTEs between 5 hours and 56 days following treatment. It was demonstrated that the manifestation of PTEs in cells treated in vitro with diethylsulphate (DES) was non-random. To investigate the potential role of higher order packaging upon the manifestation of PTEs, nuclei and DNA were isolated from cells and treated in vitro with DES. The determination of the frequency of nucleosides found 5' to PTEs revealed the same non-random manifestation of PTEs observed in the whole cells treated with DES. From these studies it would appear that neither DNA packaging nor sequence specific repair play a significant role. Studies using synthetic oligonucleotides indicate that the manifestation of PTEs may be influenced by the primary structure of DNA (i.e. its sequence). In a separate study using specifically modified olignucleotides it was demonstrated that the postlabelling assay was able to detect tandem PTE damage. However, PTEs separated by a single normal nucleotide appeared as single lesions.