STABLE TRANSFECTION IN THE MONOGENETIC TRYPANOSOMATID LEPTOMONAS-COLLOSOMA - TRANSCRIPTION BARRIER OF HETEROLOGOUS TRYPANOSOMATID SL RNA GENES AND EXPRESSION OF A CHIMERIC SL RNA MOLECULE

A stable transfection system for the lower trypanosomatid Leptomonas c ollosoma was established using the Leishmania expression vector pX tha t was derived from an extrachromosomal amplified DNA carrying the dihy drofolate 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 conc entration. The system was utilized to examine the expression of the SL RNA genes of Trypanosoma brucei and Leishmania 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 transcr iption of cells made permeable by lysolecithin, suggesting the existen ce of a transcription barrier for this gene among the trypanosomatids. Such a barrier does not exist for the T. brucei 5S rRNA gene, since t ranscription 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 the L. coll osoma SL gene was identified. Chimeric constructs carrying 50 or 415 n t of the L. collosoma SL upstream sequence and 24 nt of the L. colloso ma exon portion were fused to the T. brucei SL RNA gene at the SL port ion. Expression of a chimeric SL RNA of 150 nt, composed of 24 nt L. c ollosoma RNA and 126 nt T. brucei RNA, was observed only in cell lines carrying the 415-nt upstream sequence. The efficient expression of th e chimeric SL RNA, using the L. collosoma SL gene regulatory regions, may facilitate a structure-function analysis of chimeric and site-dire cted mutated SL RNA in trans-splicing. (C) 1996 Academic Press, Inc.