TRYPANOSOMA-CRUZI - INTERRUPTION OF BOTH ALLELES OF A GENE ENCODING APROTEIN CONTAINING 14-AMINO-ACID REPEATS BY TARGETED INSERTION OF NEO(R) AND HYG(R)

In Trypanosoma cruzi approximately 90% of the 121 and 176-kDa cytoskel etal 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 TC R27 proteins we replaced the corresponding regions of 42-nucleotide re peats 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 ana logue, and hygromycin showed that in both cases the repetitive regions had in fact been deleted. In addition, the absence of expression of t he 14-amino-acid repeats was confirmed in Western blots. In axenic cul tures growth rates of the morphologically unchanged, doubly resistant organisms were not different from those of wild-type parasites. Howeve r, 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. (C) 1995 Academic Press.