INTERRUPTION OF A TRYPANOSOMA-CRUZI GENE ENCODING A PROTEIN CONTAINING 14-AMINO ACID REPEATS BY TARGETED INSERTION OF THE NEOMYCIN PHOSPHOTRANSFERASE GENE

In Trypanosoma cruzi, the cause of Chagas' disease in Latin America, a large proportion of the antigenic proteins described to date have repetitive domains. In earlier work we identified a partial length cDNA, designated TCR27, encoding approx. 26 copies of a 14-amino acid repeat and a unique 61 -amino acid C-terminal region. The goal of the current project was to replace the repetitive region of a TCR27 gene with the neomycin phosphotransferase gene (NEO(r)). A pBluescript-based vector was constructed in which the 0.9-kb NEO(r) coding region replaced the 2.9-kb internal repetitive segment of a TCR27 gene and was in frame with its nonrepetitive 5' coding sequence (pTCR27-2=NEO). Epimastigotes were electroporated in the presence of linearized pTCR27-2=NEO and transfected clones were selected on solid medium containing G418. Southern and Northern analyses of DNAs and RNAs from four G418-resistant clones showed that in all cases the repetitive region in the smaller of the two TCR27 genes (TCR27-2) had been replaced by NEO(r). The absence of the native TCR27-2 protein in the transfected clones was confirmed by Western blot. In axenic cultures growth rates of epimastigotes bearing an interrupted TCR27-2 gene were not different from those of wild-type parasites. In addition, there was no relative impairment of the four transfected clones' ability to proliferate in cultured mammalian cells. The fact that the clones having the interrupted TCR27-2 gene were not impaired biologically suggests that the length of the repetitive region of the TCR27 protein is not a critical factor for survival.