Kinetoplast DNA replication and RNA interference in trypanosomes.

摘要

Kinetoplast DNA (kDNA) is the mitochondrial DNA of Trypanosoma brucei (the causative agent of African sleeping sickness) and related organisms. It is unique in nature and is organized into a network containing 5,000 topologically interlocked minicircles. This network, situated within the mitochondrial matrix, is condensed into a disk-shaped structure located near the basal body of the flagellum. Fluorescence in situ hybridization revealed that prior to their replication, minicircles are released vectorially from the network face nearest the flagellum. Replication initiates in the zone between the flagellar face of the disk and the mitochondrial membrane (we term this region the kinetoflagellar zone (KFZ)). The replicating minicircles then move to two antipodal sites which flank the disk-shaped network. In later stages of replication the number of free minicircles increases, accumulating transiently in the KFZ. The final replication events, including primer removal, repair of many of the gaps, and reattachment of the progeny minicircles to the network periphery, are thought to take place within the antipodal sites. To discover novel genes involved in kDNA replication we are using RNA interference (RNAi), a powerful method for inhibition of gene expression in Trypanosoma brucei. We have developed a vector (pZJM) for in vivo tetracycline-inducible synthesis of double-stranded RNA (dsRNA) in stably-transformed cells. The dsRNA is synthesized from opposing T7 promoters. We have constructed a genomic DNA-based RNAi library using this vector and are now in process of using this as a forward genetic strategy.