Rice Genetics IV - IRRI books - International Rice Research Institute

Level 2: activation of local repair complexesMost of the transgenic plants we have analyzed have only a single transgenic locus asdefined by genetic segregation analysis. However, as discussed above, individualtransgenes and contiguous transgene arrays have been shown to be arranged in a localcluster, also containing significant stretches of genomic DNA. This is not the filler DNAdescribed above, which is usually only a few nucleotides in length.The presence of significant stretches of genomic DNA within a transgene clustersuggests that there is a pronounced tendency for fragments of exogenous DNA to integrateat one site. Since transgenic loci appear to arise randomly, probably reflectingnaturally occurring DNA breaks, we proposed that a primary integration event promotedsecondary integration events nearby (Kohli et al 1998). This two-phase mechanismcould reflect the recruitment of DNA-repair complexes to the original integrationsite and the consequent introduction of additional double-strand DNA breaks in thelocal region. This mechanism would result in a concentrated core of DNA repair andtransgene integration, which would likely cause a significant amount of transgene rearrangementas well as the loss of stretches of genomic DNA. Indeed, the deletion ofgenomic DNA around transgene integration sites is a well-known phenomenon in bothtransgenic plants and animals.Level 3: integration at the chromatin levelThe third level of transgene organization involves the dispersion of transgene copiesand clusters such that individual signals can be visualized by FISH on metaphasechromosomes. This demonstrates that transgene clusters may be separated bymegabases of DNA. How is this different from the level 2 organization discussedabove? Could it be that repair complexes are being activated over a greater area togenerate transgene sites separated by larger intervening regions of genomic DNA?Two pieces of evidence suggest not. First, the intervening regions at level 2 organizationcan be isolated by long PCR, a reasonable distance to be spanned by locallyrecruited repair complexes. However, repair complexes would unlikely be recruitedto sites megabases away from the initial integration site. Second, the analysis of thesame wheat nuclei at interphase shows the remarkable phenomenon that the FISHsignals that are separated at metaphase are brought back together at interphase(Abranches et al, n.d.). Since this phenomenon occurs in multiple nuclei from somatictissues of a given transgenic plant, and in progeny thereof, it confirms that theinterphase chromatin is highly and reproducibly organized in the nucleus. We haveput forward three models to explain this (Abranches et al, n.d.). First, it is possiblethat the homologous DNA sequences are associating in trans. Second, copies of thesame promoter may be recruited to a common transcription factory in the nucleus.Our third model, which provokes the most thought with respect to the mechanisms oftransgene integration, is that the FISH signals permit visualization of the three-dimensionalconfiguration of the nucleus at the moment of transformation. It has beenshown that cells successfully transformed by particle bombardment usually have themetal particle lodged in the nucleus. Therefore, the metal particle may causelocalized damage to DNA in a particular region of the nucleus. The way that chroma-Transgene integration, organization, and expression . . . 457