Trypanosoma brucei: improved detection of nuclear transcripts reveals a genomic position effect on nuclearly...

Köhler, S. 1999. Trypanosoma brucei: Improved detection of nuclear transcripts reveals a genomic position effect on nuclearly accumulating NEO RNAs visualized in stably transformed cells. Experimental Parasitology 92, 249-262. An improved fluorescent in situ hybridization method was used to visualize accumulations of nuclear RNA in procyclic Trypanosoma brucei that were stably transformed with a bacterial gene encoding neomycin phosphotransferase (NEO). In both wild-type trypanosomes and NEO transformants, nuclear accumulations of endogenous RRNAs were restricted to a ring-shaped subcompartment (nucleolus) of the nucleus. A nucleolar localization was also illuminated for NEO RNAs of trypanosomal transformants mediating their NEO transcription from the endogenous RRNA gene cluster of the T. brucei genome. In contrast, stably transformed trypanosomes generating their NEO transcripts from protein-coding areas of the T. brucei genome displayed a single dot-like accumulation of NEO transcripts, which was located in close proximity to the trypanosomal nucleolus. This pattern was observed in transformants employing either the promoter region of a protein-coding PARP transcription unit or the trypanosomal RRNA promoter for NEO transcription. Apparently, the exact location of nuclearly accumulating NEO transcripts varied among different trypanosomal transformants and relied explicitly on the genomic position of the NEO gene. These results imply that T. brucei possesses distinctive pathways for its nuclear RNA metabolism, which is consistent with a spatio-functional organization of the parasite's nucleus.