The tandemly-arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 being expressed predominantly in metacyclics whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.8 (typical of CPB3-CPB18) revealed two major regions of divergence: one of 258 bp corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totalling 57 bp, localised to the intercistronic region downstream of CPB1 and CPB2. Cell lines expressing CPB2.8 or CPB2 with the 3'-UTR and intercistronic sequence of CPB2.8 showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred with CPB2 or CPB2.8 with the 3'-UTR and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclic-specific, stage-regulated expression of CPB by affecting the maturation of polycistronic pre-mRNA. This is the first well-defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania
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