In vitro transcription in isolated nucleoli of Tetrahymena pyriformis

An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5' region of their 35S pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of transcriptional control during development and differentiation.