During their life cycle Trypanosoma cruzi parasites multiply as extracellular epimastigotes in the reduviid bug and as intracellular amastigotes in the mammalian host. The genome of T. cruzi contains a cluster of tandemly arrayed, alternating genes encoding proteins called amastin and tuzin. Amastin is an abundant protein on the surface of amastigotes and its mRNA constitutes 1-2% of the total mRNA at this stage. Tuzin is a less abundant protein of unknown cellular location. The amastin/tuzin gene cluster is constitutively transcribed in epimastigotes and amastigotes, but the steady state level of amastin mRNA is 85-fold higher in amastigotes than in epimastigotes. The 3' untranslated region of the amastin gene is involved in this differential expression. Tuzin mRNA levels do not differ substantively in the two life stages. We propose to elucidate the posttranscriptional events that enhance expression of the amastin genes in amastigotes and maintain a relatively constant level of expression of the tuzin genes in both amastigotes and epimastigotes. A combination of linker replacements and protein binding assays will be used in these studies. In addition, we plan to utilize the sequence of the amastin 3' untranslated region and downstream intergenic region to differentially express in amastigotes specific proteins that will be used to (i) develop a more sensitive detection assay for amastigotes in T. cruzi- infected animals, (ii) examine whether animals already infected with T. cruzi are less susceptible to challenge by T. cruzi than are uninfected animals, (iii) determine the cellular location of tuzin, and (iv) induce amastigotes to undergo programmed cell death. Finally, we will assess whether amastin and/or tuzin can serve a target antigens for an improved diagnostic test for T. cruzi infection.
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