COLOGNE, GERMANY

Max-Planck-Institut

Integration of dSpm in transgenic tobacco

--Guillermo Cardon, Julio Reinecke, Monika Frey, Heinz Saedler and Alfons Gierl

An excision assay system was recently established in transgenic tobacco (Frey et al., EMBO J. 9:4037-4044, 1990) for the En/Spm transposable element. In this model system two En/Spm encoded functions, TNPA and TNPD, are required for excision of a dSpm reporter element. TNPA is the product of the most abundant 2.6kb mRNA that has been previously identified as the suppressor component of En/Spm (Grant et al., EMBO J. 9:2029-9035). TNPD is the product of the less abundant 5.9kb mRNA (Masson et al., Cell 58:755-765, 1989). The sequence alterations (footprints) generated during excision are similar to the ones observed in maize. This suggests that the mechanism of excision promoted by TNPA and TNPD is similar, if not identical, to the one existing in maize. Based on these results a model for En/Spm excision was proposed (Frey et al., 1990): Binding of TNPA to the subterminal region of the element induces complex formation between both ends by a “zipper”-like mechanism, where the bound TNPA molecules represent the teeth of the zipper. This leads to “correct” alignment of the terminal inverted repeats (TIRs). Binding of TNPD to the TIRs leads to endonucleolytic cleavage and release of En/Spm from the site of integration accompanied by healing of the chromosome breaks.

In order to analyse transposition in this transgenic system, four independent dSpm integration events were molecularly cloned into the lambda EMBL4 vector. All clones represent completely transposed dSpm elements, and their insertion generated the characteristic three base pair target site duplication. Therefore, the mechanism of integration in the presence of TNPA and TNPD in the transgenic system seems to be identical to the one present in maize. Three of the transposed dSpm elements moved into single copy DNA that is relatively undermethylated, as judged by methylation sensitive restriction enzymes. One of the transposed dSpm elements moved into middle repetitive, methylated DNA. The regions surrounding the new insertion sites do not show any significant homology to En/Spm specific sequences. These results suggest that this system might be suitable for gene tagging in transgenic systems.


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