A foreign transcription unit in the inactivated VSG gene expression site of the procyclic form of Trypanosoma brucei and formation of large episomes in stably transformed trypanosomes

Abstract

Transcription of the variant surface glycoprotein (VSG) gene expression site (ES) in Trypanosoma brucei is inactivated upon differentiation from the bloodstream form to the insect-adapted, procyclic form. This paper demonstrates that a foreign transcription unit, containing a procyclic acidic repetitive protein (PARP) gene promoter driving a neomycin phosphotransferase (neo) gene, can be fully active once integrated at the lingering, transcriptionally inactive VSG ES of procyclic trypanosomes. Following targeting into the ES two types of transformants were identified. Type one transformants were generated by integration of the PARP-neo gene into the region downstream of the long 70-bp repeat array of the silent telomeric ES encoding VSG gene 118. alpha-amanitin-resistant transcription at the neo locus proceeded from the PARP promoter to approximately 2.5 kb downstream of the integration site and terminated in front of the VSG 118 gene. Type two transformants contained variously sized large episomes (ranging from 135 kb to 500 kb), consisting of tandemly linked input plasmids. Transcription of the neo gene in the episomes was also resistant to alpha-amanitin. The presence of large amounts of the active episomal PARP promoter did not significantly affect the transcription of most RNA polymerase II transcribed genes, but resulted in a significant and equal reduction of the transcriptional efficiency of the endogenous PARP genes and VSG gene promoter sequences. This observation suggests that transcription of the PARP gene and the VSG gene expression sites in insect form trypanosomes may share a common transcriptional machinery.