IN-VITRO TRANSCRIPTION OF THE LEPTOMONAS-SEYMOURI SL RNA AND U2 SNRNAGENES USING HOMOLOGOUS CELL-EXTRACTS

A cell-free transcription system for the spliced leader (SL) RNA gene of the trypanosomatid Leptomonas seymouri: has been developed. Accurat ely initiated transcription was achieved using cell extracts and a tem plate in which the transcribed region of the SL RNA was replaced with a guanosine-less sequence (G-less cassette). The extract was also able to direct accurate initiation of RNA from an L. seymouri tagged U2 sn RNA gene, which may be expressed via a transcriptional apparatus share d by the SL RNA gene. In vivo transcription analysis was used previous ly to define essential sequence components of the SL RNA gene promoter (Hartree D, Bellofatto V. Mol Biochem Parasitol 1995;71:27-39). A sub stitution mutation in the upstream promoter element (bp -50 to -70) ma rkedly reduced transcription in vitro as did deletion of this and the middle promoter element (bp -30 to -40). Thus, the in vitro transcript ion system correctly responds to promoter mutations and is useful for investigating SL RNA and snRNA gene expression. (C) 1997 Elsevier Scie nce B.V.