THE REGION ENCOMPASSING THE PROCYCLIC ACIDIC REPETITIVE PROTEIN (PARP) GENE PROMOTER PLAYS A ROLE IN PLASMID DNA-REPLICATION IN TRYPANOSOMA-BRUCEI

We have previously reported the construction and characterization of a n autonomously replicating plasmid in Trypanosoma brucei. In this plas mid the procyclic acidic repetitive protein (PARP) gene promoter drive s the transcription of a selectable marker. Deletion of this promoter incapacitates the plasmid, suggesting its utilization as a promoter-tr ap. Three independent libraries were created by inserting variously di gested T.brucei genomic DNA into this promoterless construct. Transfec tion of these libraries into procyclic T.brucei and the subsequent iso lation of episomes led only to the reisolation of the PARP promoter. A dditionally, a ribosomal RNA promoter failed to keep the construct as an episome, although it can sustain mRNA transcription in T.brucei and was shown to be an efficient promoter in this construct. Finally, by using a transient replication assay involving the methylation-sensitiv e restriction endonuclease DpnI to distinguish between input and repli cated DNA, we showed that the PARP promoter-bearing construct could re plicate autonomously in procyclic T.brucei, but the corresponding cons truct with the rRNA promoter could not. The close association between elements that sustain transcription and DNA replication in T.brucei mi rrors results observed in several higher eukaryotes and their viruses and suggests an ancient origin of this feature.