STAGE-INDEPENDENT SPLICING OF TRANSCRIPTS FROM 2 HETEROGENEOUS NEIGHBORING GENES IN LEISHMANIA-AMAZONENSIS

Gene expression in trypanosomatid protozoa is largely regulated posttr anscriptionally, e.g., 5' splice leader addition and 3' polyadenylatio n of mRNAs. We examined these events in Leishmania by mapping the spli ce sites of the transcripts from two different, but closely linked sin gle-copy genes 2.3 kb apart. The coding regions of the approx. 1 kb up stream gene (P36) and the approx. 1.4 kb downstream gene (NAGT) produc e approx. 2 and 3 kb mRNAs, respectively. Both genes were overexpresse d in cells that were transfected with this bicistronic unit (greater t han or equal to 7.5 kb), taking advantage of the NAGT as a selectable marker for tunicamycin-resistance. The transcripts from both genes wer e spliced constitutively at bo th ends, irrespective of their episomal or chromosomal expression in both leishmanial stages. Primer extensio n of the 5' UTRs and S1 nuclease protection of the 3' UTRs initially i dentified the major splice sites, corresponding to the genomic sequenc e at -205 bp and + approx. 900 bp of P36, and -1012 bp and + approx. 6 00 bp of NAGT. These splice sites, consistent with the size of the maj or transcripts, are among those mapped precisely by sequencing RT-PCR amplified 5' and 3' UTRs. The additional sites mapped by the latter ar e minor alternatives, especially abundant for transcripts of the downs tream NAGT. All these minor splice sites are closer than the major spl ice sites to the coding region, indicating that the most distant splic e sites are preferentially used. This preference creates a 387 bp 'gap ' with polypyrimidine tracts in the intergenic region consistent with the model coupling splice leader addition with polyadenylation in pre- mRNA processing. The stage-independence of these events suggests that the 7.5 kb dicistronic unit is suitable for constructing Leishmania-sp ecific constitutive expression vectors. (C) 1997 Elsevier Science B.V.