FIGURE 2.
Clitocine is incorporated into RNA in the position of adenosine. (A) Phosphorimager analysis showing that T7 RNA polymerase transcription of a pre-tRNATyr cDNA labeled with UTP-[α-32P] or CTP-[α-32P] produces full-length transcripts when all four cognate nucleotide triphosphates are present or when ATP is substituted with clitocine-triphosphate. Abortive products are seen in lanes 9 and 10 because the small amount of UTP-[α-32P] or CTP-[α-32P] allows some transcription without UTP or CTP, respectively. (B) Phosphorimager analysis of 2D TLC showing that clitocine is present in in-vitro-transcribed RNAs that were labeled with UTP-[α-32P] (as in panel A), digested with T2 RNase, and separated by 2D TLC. Panels are labeled with nucleotides included in the T7 transcription reactions. A/cl indicates that an equal molar ratio of ATP and clitocine-TP was used. TLC spots corresponding to the monophosphate nucleotides, Ap (adenosine-3′-phosphate), Cp (cytosine-3′-phosphate), Gp (guanidine-3′-phosphate), Up (uridine-3′-phosphate), and clp (clitocine-3′-phosphate), are indicated (Bochner and Ames 1982). The results provided a 2D TLC position reference for clitocine-3′-p.