Stage-regulated expression of cruzipain, the major cysteine protease of Trypanosoma cruzi is independent of the level of RNA

The genes that encode cruzipain, the major cysteine protease of Trypanosoma cruzi are known to be arranged in tandem arrays. To gain a detailed insight into how these arrays are organised at the chromosomal level we have isolated clones from a cosmid library constructed with DNA from the X10.6 strain. In this strain we found that cruzipain is encoded by two allelic clusters composed of approximately 14 and 23 tandemly repeated genes which are located on homologous chromosomes of 650 and 670 kb. With the exception of the 3'-proximal genes, the cruzipain genes were all of identical or very similar sequence. An unusual feature of the 3'-proximal genes is that they lack the sequences that encode the 130 amino acid carboxyl terminal extension which is characteristic of cruzipain. Both gene clusters are situated in a similar chromosomal environment and are flanked by sequences which have the potential to form Z-DNA. In other eukaryotes, these motifs have been associated with recombinational hotspots and have been demonstrated to enhance gene conversion. The cruzipain genes are transcribed to produce a 1.8-kb transcript which is present at the same steady-state level in each of the parasite life cycle stages. However, protein levels and activity are 4-5-times higher in the insect epimastigote stage than in the trypomastigote and amastigote stages. By implication developmental regulation of cruzipain expression occurs predominantly at the translational and/or post-translational levels.