Analysis of a hybrid PARP/VSG ES 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.
Qi
C C
CC
Department of Medicine, Columbia College of Physicians and Surgeons, New York, NY 10032, USA.
Urményi
T
T
Gottesdiener
K M
KM
D000546:Amanitins / Q000494:pharmacology; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*; D012319:RNA Polymerase II / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D016053:RNA, Protozoan / Q000032:analysis / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000254:growth & development; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000235:genetics*
D000546:Amanitins; D016054:DNA, Protozoan; D004274:DNA, Recombinant; D008562:Membrane Glycoproteins; D019384:Nucleic Acid Synthesis Inhibitors; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D014643:Variant Surface Glycoproteins, Trypanosoma; C053277:procyclic acidic repetitive protein, Trypanosoma; D015500:Chloramphenicol O-Acetyltransferase; D012319:RNA Polymerase II