Analysis of a hybrid [formula omitted] promoter in procyclic trypanosomes

Abstract

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 Ürményi and Van der Ploeg (Ürményi, 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 α-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.