Gene expression in Leishmania: Analysis of essential 5′ DNA sequences

A major unanswered question in Kinetoplas parasites is the mechanism of regulating gene expression. ing a transfection system, we have previously shown that the genic region of the α-tubulin gene of Leishmania enriettii ned sequences required for gene expression. The goal of work reported here was to determine whether the Leishmania-derived sequences were providing transcriptional con signals or functioning at a post-transcriptional level, most in trans-splicing. The chloramphenicol acetyltransferase gene was used as the reporter gene and was stably educed into L. enriettii as part of an extrachromosomal ent by transfection. We show here that the production of mRNA was dramatically dependent on the presence of the genic region 5′ to the cat gene. The intergenic region could substituted by a smaller fragment (222 base pairs) that ined the trans-splice acceptor site and an adjacent poly idine tract. This native fragment could be replaced by a etic polypyrimidine tract containing an AG site. The ve and the synthetic fragments had unidirectional activity. effect on transcription of the cat gene by the wild-type ent or the synthetic polypyrimidine was detected. The ts indicate that both regions contain signals that affect DNA stability, probably sequences involved in trans-splicing.