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Nucleic Acids Research, 1990, Vol. 18, No. 11 3319-3326
© 1990

MOLECULAR BIOLOGY

Permeable trypanosome cells as a model system for transceiption and trans-splicing

Elisabette Ullu1,2,* and Christian Tschudi1

1Yale MacArthur Center for Molecular Parasitology, Department of Internal Medicine 333 Cedar Street, New haven, CT 06510, USA 2Cell Biology, Yale University School of Medicine 333 Cedar Street, New haven, CT 06510, USA

*To whom correspondence should be addressed

Received February 2, 1990. Accepted April 19, 1990.

We have established conditions for Trypanosoma brucei permeable cells to study transcription and transsplicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCI, whereas higher potassium concentrations were inhibitory. In addition, MgCL2 concentrations above 3 mM led to the accumulation of a 3' end shortened SL RNA species, which has been previously reported not to participate in trans-splicing (20). Using conditions optimal for the synthesis of the SL RNA, we observed accurate transsplicing of newly-synthesized {alpha}-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced {alpha}-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled.


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