Functional analysis of the intergenic regions of TcP2β gene loci allowed the construction of an improved Trypanosoma cruzi expression vector

• Published in: Gene 1999-11, Vol.239 (2), p.217-225
• Main Authors: Vazquez, Martin P, Levin, Mariano J
• Format: Article
• Language: English
• Subjects: Expression plasmid; Overexpression; Repetitive elements; SIRE; Trypanosoma cruzi
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(99)00386-8

TcP2β ribosomal protein genes in Trypanosoma cruzi are encoded by four different loci, H6.4, H1.8, H1.5 and H1.3. All loci have a similar organization, except for H1.8 that harbors two TcP2β genes arranged in tandem and separated by a short repetitive sequence, named SIRE (short interspersed repetitive element), which is also found upstream of the first gene of the tandem and downstream of the second. In this locus the trans-splicing signal of TcP2β is located within the SIRE element, while in the other loci it is positioned within the first 50 bases upstream of the AUG with an AG acceptor site at position −12 respective to the initiation codon. Transient transfection experiments were used to evaluate the efficiency of these two different trans-splicing regions to drive CAT activity. The region named HX1 located upstream the TcP2β H1.8 gene was clearly more efficient than the SIRE sequence contained in the region named HX2. Therefore, we decided to use the HX1 region to ameliorate the performance of the cryptic trans-splicing signal present in the T. cruzi expression vector pRIBOTEX (Martinez-Calvillo, S., López, I., Hernandez, H., 1997. pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants. Gene 199, 71–76). By insertion of the region HX1 downstream of the ribosomal promoter of pRIBOTEX, we constructed pRHX1CAT40 that, in stable transfected cells, was able to drive CAT activity 2760 times more efficiently than the control plasmids. Based on this, a novel plasmid vector was conceived, named pTREX-n, which retains the neo gene of pRIBOTEX as a positive selectable marker and replaces the CAT-SV40 cassette in pRHX1CAT40 by a multiple cloning site.