Cloning, characterization and developmental gene regulation of the flagellar gene, PFR-2, in Leishmania mexicana

Protozoan parasites of the genus Leishmania have evolved a complex life cycle involving two divergent host organisms. Within each respective host, specific morphological and biochemical changes are required. However, little is known about how Leishmania regulate their genes. This study sought to identify developmentally regulated genes and study the mechanism of their developmental gene regulation. Flagellar genes were characterized since the flagellum is developmentally controlled. Complex polyclonal antisera raised against total flagella protein was used to screen a promastigote cDNA expression library. Positive cDNA clones were subjected to Northern analysis to identify candidates for developmentally regulated mRNAs. Two cDNAs, fla2 and fla8, were identified as encoding developmentally regulated, flagellar genes. These two cDNAs encode the two major paraflagellar rod proteins in Leishmania mexicana, PFR-2 and PFR-1. The gene(s) encoding PFR-2 were cloned and found to be present in the genome as a tandem array of three genes that are transcribed polycistronically along with two upstream and two downstream transcripts. Transcription of the PFR-2 locus is not decreased in amastigote nuclei indicating that developmental regulation is post-transcriptional. Transient transfection and stabe transfection experiments indicate that the downstream intergenic region separating PFR-2C from the downstream transcript, D1, confers a 3.3-fold developmental gene regulation. This is similar to developmental regulation of the D1 transcript suggesting that a cis-acting element in the PFR-2C-D1 intergenic region coordinately controls both upstream and downstream transcripts. This control of gene expression is most likely due to control of RNA processing since both trans-splicing of the downstream gene and polyadenylation of the upstream gene are coupled processes