Stable expression of the bacterial neo^r gene in Leishmania enriettii

Abstract

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^1,2. We now describe the stable expression of a selectable marker, the gene for neomycin resistance (neo^r) in Leishmania enriettii. A chimaeric gene containing the neo^r gene inserted between two α-tubulin intergenic sequences was introduced into the cells and drug-resistant L. enriettii were observed which stably expressed theneo^r gene. One goal of this work was to analyse the sequences necessary fortrans-splicing of messenger RNA, as try-panosomatids have a novel process of RNA trans-splicing, described initially in Trypanosome brucei^3–5 and subsequently in several other trypanosomatids, including L. enriettii⁶. Many try-panosomatid 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 site6. Messenger RNA isolated from several differentneo^r L. enrietii lines contained the spliced leader sequence joined to the neo^r gene at the position of the splice acceptor site in the α-tubulin intergenic sequence. Theneo^r 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.