Cloning and characterization of the metacyclogenin gene, which is specifically expressed during Trypanosoma cruzi metacyclogenesis

We isolated a gene that is differentially expressed during Trypanosoma cruz i metacyclogenesis by the representation of differential expression (RDE) m ethod. using differentiating epimastigotes cultured in chemically defined m edium. This gene, the metacyclogenin gene, encodes a 630-nucleotide mRNA th at is specifically associated with the polysomes of epimastigotes allowed t o differentiate for 24 h. We sequenced and characterized the metacyclogenin gene and found that there were at least three copies of the gene organized into tandem 2.8 kb repeats in the genome of T. cruzi Dm28c. We analyzed th e repeats and found that they contained two other genes, one encoding trypa redoxin peroxidase and the other encoding a 0.6 kb mRNA (named associated g ene or AG) with sequences showing no significant similarity to those in the GenBank database. Northern blot analysis of polysomal RNA extracted from r eplicating and differentiating epimastigotes showed that metacyclogenin and AG genes displayed similar patterns of expression. Their products were det ected only in differentiating epimastigotes, whereas tryparedoxin peroxidas e was detected only in the polysomal RNA fraction of replicating and differ entiating epimastigotes. In Northern blots of total RNA from differentiatin g and replicating epimastigotes. the genes studied were detected in both ce ll populations. The differential expression of the metacyclogenin gene was confirmed by immunocytochemistry studies showing that the protein is detect ed only in differentiating (adhered) epimastigote. The results suggest that mRNA mobilization to polysomes is an important mechanism in the regulation of gene expression in T. cruzi. (C) 2001 Elsevier Science B.V. All rights reserved.