A stable transfection system for the lower trypanosomatid Leptomonas collosoma was established using the Leishmania expression 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 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 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 the T. brucei 5S 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 the L. collosoma SL gene was identified. Chimeric constructs carrying 50 or 415 nt of the L. collosoma SL upstream sequence and 24 nt of the L. collosoma exon portion were fused to the T. brucei SL RNA gene at the SL portion. Expression of a chimeric SL RNA of 150 nt, composed of 24 nt L. collosoma RNA and 126 nt T brucei RNA, was observed only in cell lines carrying the 415-nt upstream sequence. The efficient expression of the chimeric SL RNA, using the L. collosoma SL gene regulatory regions may facilitate a structure-function analysis of chimeric and site-directed mutated SL RNA in trans-splicing.
Bibliographical noteFunding Information:
We thank Dr. Stephen M. Beverley for providing the pX plasmid, Dr. Michal Shapira for the Leishmania RNA, Dr. Nina Agabian for the T. brucei SL gene, Sara Weiss Feigelson and Michal Safran for helping with the pX-derived constructs, and Daniel Klebanov for the art work. This work was supported by a grant from the Catherine T. MacArthur Foundation to the Weizmann Institute and by a grant from the Leo and Julia Forchheimer Center for Molecular Genetics at the Weizmann Institute of Science.