Stable expression of the bacterial neor gene in Leishmania enriettii

• Published in: Nature (London) 1990-02, Vol.343 (6258), p.572-574
• Main Authors: Laban, Avraham, Tobin, James Finbarr, de Lafaille, Maria A. Curotto, Wirth, Dyann F.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Chromosomes; Cloning, Molecular; Deoxyribonucleic acid; DNA; DNA Probes; Drug resistance; Drug Resistance, Microbial - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Gene sequencing; Genes; Genes, Bacterial; Genetic Markers; Humanities and Social Sciences; Hybridization; Leishmania mexicana - genetics; letter; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; mRNA; multidisciplinary; Neomycin; NeoR gene; Nucleic Acid Hybridization; Nucleotide sequence; Parasites; Plasmids; Polyadenine; Polyadenylation; Protozoa; Restriction Mapping; RNA Splicing; RNA, Messenger - genetics; Science; Science (multidisciplinary); Splicing; Transfection; Tubulin; Tubulin - genetics
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/343572a0

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 the neo r gene. One goal of this work was to analyse the sequences necessary for trans -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. enriettii 6 . 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 different neo 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. The neo 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.