Assessing the effectiveness of coding and non-coding regions in antisense ribosome inhibition of gene expression in Tetrahymena

In Tetrahymena thermophila, an "antisense ribosome" technology has been developed for inhibiting gene expression and generating novel mutants. Short segments of genes are inserted in antisense orientation into an rDNA vector in a region corresponding to an external loop of the folded rRNA. DNA segments derived from the 5'-ends of genes have proven most effective in reducing cognate gene expression. To investigate the efficacy of other genic regions, we generated Tetrahymena cell lines with antisense ribosome constructs containing 100-bp DNA segments derived from the 5'-ends, 3'-ends, and internal coding regions of two nonessential genes, granule lattice protein 1 and macronuclear historic H1. The 5'- and 3'-end constructs inhibited gene expression, but antisense ribosomes derived exclusively from coding regions had little effect.