Trypanosoma cruzi: Interruption of Both Alleles of a Gene Encoding a Protein Containing 14-Amino-Acid Repeats by Targeted Insertion of NEOr and HYGr

Abstract

In Trypanosoma cruzi approximately 90% of the 121 and 176-kDa cytoskeletal proteins encoded by the two alleles Of the TCR27 gene is composed of 14-amino-acid repeats. To gain insight into the function of the TCR27 proteins we replaced the corresponding regions of 42-nucleotide repeats in the two alleles with the NEO r and HYG r genes. Analyses of DNAs and RNAs from four clones resistant to both G418, a neomycin analogue, and hygromycin showed that in both cases the repetitive regions had in fact been deleted. In addition, the absence of expression of the 14-amino-acid repeats was confirmed in Western blots. In axenic cultures growth rates of the morphologically unchanged, doubly resistant organisms were not different from those of wild-type parasites. However, the doubly resistant organisms proliferated more slowly in cultured mammalian cells than did wild-type parasites. These findings indicate that the absence of the TCR27 repetitive regions is detrimental, but not fatal, to the parasites.