Integration of expression vectors into the ribosomal locus of Trypanosoma cruzi

Abstract

The expression vectors of the protozoan parasite Trypanosoma cruzi pRIBOTEX and pTREX harbor a ribosomal promoter that improves gene expression and clone selection. Interestingly, the solely presence of this 810 bp long sequence leads to the integration of these vectors into the ribosomal locus, even though circular plasmids are poorly recombinogenic. Initially, it was suggested that a 174 bp long ribosomal-specific repeat element present in the ribosomal promoter region could be responsible for the genetic exchange. On the contrary, we demonstrate that recombination of pTREX occurs within a 86 bp long region located 120 bp downstream the transcription start point (tsp1) of the ribosomal promoter, and it does not depend on the presence of the ribosomal repeat. We also determined that a 291 bp segment encompassing the tsp1 and the 86 bp long recombination region contains all necessary signals to drive transcription and complete recombination into the rRNA locus. Finally, we demonstrate that the integration of pTREX derived plasmids into the nuclear genome occurs within the first 5 h post-transfection, and that non-integrated copies are rapidly degraded.