Identification and sequencing of two isopentenyladenosine-modified transfer RNAs from Chinese hamster ovary cells

To determine the presence and identity of isopentenyladenosine-containing t ransfer RNAs (tRNAs) in a mammalian cell line, we adopted a novel method to isolate, clone and sequence these RNAs, This method was based on 3' polyad enylation of the tRNA prior to cDNA synthesis, PCR amplification, cloning a nd DNA sequencing. Using this unique procedure, we report the cloning and s equencing of the selenocysteine-tRNA and mitochondrial tryptophan-tRNA from Chinese hamster ovary cells which contain this specific tRNA modification. This new method will be useful in the identification of other tRNAs and ot her small RNAs where the primary sequence is unknown.