Analysis of a hybrid promoter in procyclic trypanosomes

The parasite Trypanosoma brucei changes its variant surface glycoprotein (VSG) coat to escape the host immune system. At a chromosomal locus, we analyzed the promoter that controls expression of VSG genes, using a system developed in collaboration with Urményi and Van der Ploeg (Urményi, T.P. and Van der Ploeg, L.H.T. (1995) Nucleic Acids Res. 23,1010-1016), and showed that the variant surface glycoprotein expression site (VSG ES) promoter directed < 6% the CAT activity produced by the procyclic acidic repetitive protein (PARP) promoter at the same locus. We identified a fragment from the PARP promoter (bp -743 to -111) that contained no intrinsic promoter activity. However, when this fragment was cloned 5' to 3' upstream of the VSG ES promoter, and this hybrid PARP/VSG ES promoter was stably integrated at the RNA polymerase (Pol) II largest subunit gene locus, expression from a CAT gene cassette increased 10-fold. Nascent RNA analysis independently showed that the relative efficiency of alpha-amanitin-resistant transcription directed by the hybrid PARP/VSG ES promoter was more than 6-fold higher than that directed by the wild-type VSG ES promoter. Furthermore, using nascent RNA protection assays, we mapped the transcription start site of the hybrid PARP/VSG ES promoter to the same initiation site as that of the wild-type VSG ES promoter. Finally, we evaluated the functional activity of the hybrid PARP/VSG ES mutant promoter at the dominant VSG gene expression site on the 1.5-Mb chromosome. At this locus, as well, the hybrid PARP/VSG ES promoter directed almost 3-times as much CAT activity as that of the wild-type VSG ES promoter.