Stable Transfection in the Monogenetic TrypanosomatidLeptomonas collosoma—Transcription Barrier of Heterologous Trypanosomatid SL RNA Genes and Expression of a Chimeric SL RNA Molecule

Abstract

GOLDRING, A., ZIMMER, Y., BEN-YEHUDA, E., GONCHAROV, I., and MICHAELI, S. 1996. Stable transfection in the monogenetic trypanosomatidLeptomonas collosoma—Transcription barrier of heterologous trypanosomatid SL RNA genes and expression of a chimeric SL RNA molecule.Experimental Parasitology84,28–41. A stable transfection system for the lower trypanosomatidLeptomonas collosomawas established using theLeishmaniaexpression vector pX that was derived from an extrachromosomal amplified DNA carrying the dihydrofolate reductase–thymidylate synthase gene. Transformants harboring the pX vector were selected on Geneticin, and cell lines harboring as many as 200 copies per cell were obtained by increasing the drug concentration. The system was utilized to examine the expression of the SL RNA genes ofTrypanosoma bruceiandLeishmania mexicana amazonensis.Despite the high copy number of the foreign genes, no heterologous SL RNA was detected in steady-state RNA populations or by nascent transcription of cells made permeable by lysolecithin, suggesting the existence of a transcription barrier for this gene among the trypanosomatids. Such a barrier does not exist for theT. brucei5S rRNA gene, since transcription of this gene was detected in permeable cells carrying the heterologous gene and in steady-state RNA population. To overcome the transcription barrier, the authentic regulatory region of theL. collosomaSL gene was identified. Chimeric constructs carrying 50 or 415 nt of theL. collosomaSL upstream sequence and 24 nt of theL. collosomaexon portion were fused to theT. bruceiSL RNA gene at the SL portion. Expression of a chimeric SL RNA of 150 nt, composed of 24 ntL. collosomaRNA and 126 ntT. bruceiRNA, was observed only in cell lines carrying the 415-nt upstream sequence. The efficient expression of the chimeric SL RNA, using theL. collosomaSL gene regulatory regions, may facilitate a structure–function analysis of chimeric and site-directed mutated SL RNA intrans-splicing.