Organizational complexity of a rice transgene locus susceptible to methylation-based silencing

Molecular analyses of a rice (Oryza sativa L.) transgene locus introduced using biolistic techniques revealed the presence of multiple copies of rearranged fragments, as well as an intact copy of the supplied constructs. Both the gene of interest (35S-Btt cryIIIA) and the selectable marker used (Ubi1-bar) were methylated and silenced. Additionally, vector sequences were present in great abundance and were also highly methylated, indicating that the entire transgene insert was marked for methylation. The rearrangement of input DNA resulted in interspersion of plasmid backbone regions with the gene of interest. Permutation of segments encoding the gene of interest and the selectable marker was also detected, perhaps explaining why sequences introduced on separate plasmids are frequently found to be inserted at the same locus. The 35S promoter contained several hotspots for fragmentation. These observations strongly support the concept that intrusive DNA is recognized by host surveillance systems and that transgene loci with anomalous structural organization are subjected to inactivation by processes such as methylation.