Stable expression of the bacterial neor gene in Leishmania enriettii

Molecular genetic studies in parasitic protozoa have been hindered by the lack of methods for the introduction and expression of modified or foreign genes in these organisms. Two recent reports described the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of parasite-specific sequences. We now describe the stable expression of a selectable marker, the gene for neomycin resistance (neor) in Leishmania enriettii. A chimaeric gene containing the neor gene inserted between two alpha-tubulin intergenic sequences was introduced into the cells and drug-resistant L. enriettii were observed which stably expressed the neor gene. One goal of this work was to analyse the sequences necessary for trans-splicing of messenger RNA, as trypanosomatids have a novel process of RNA trans-splicing, described initially in Trypanosome brucei and subsequently in several other trypanosomatids, including L. enriettii. Many trypanosomatid genes are arranged in tandem arrays and the intergenic sequences contain both the splice acceptor site for the addition of the spliced leader sequence and a putative polyadenylation site. Messenger RNA isolated from several different neor L. enrietti lines contained the spliced leader sequence joined to the neor gene at the position of the splice acceptor site in the alpha-tubulin intergenic sequence. The neor mRNA was also polyadenylated. Plasmid DNA is present within the drug-resistant organisms and appears to be extrachromosomal. The development of these methods allows the functional analysis of sequences necessary for trans-splicing.