Figure 4.

(A) tRNA-specific adenosine deaminase assay. ³³P-labeled alanine tRNAs (200 fmol) from B. mori (B.m.) and yeast (y) were incubated for 1 h with purified, recombinant hADAT1 and analyzed by one-dimensional TLC after P1 nuclease digestion of the processed RNA. Lane 1, no protein; lanes 2–6, 1.5 μl of hADAT1. A34G, mutant tRNA containing G₃₄ instead of A₃₄; A37G, tRNA with A₃₇ to G₃₇ mutation. The spots at the bottom are the origin; the spots in the middle correspond to AMP; the spots at the top correspond to IMP. (B) Sequence analysis of in vitro-assayed human tRNA^Ala. In vitro-transcribed human tRNA^Ala was incubated with 2 μl of recombinant, purified human ADAT1 (hADAT1) or with buffer (control). Human tRNA^Ala-specific reverse transcription–PCR products were sequenced. Only the anticodon loop-region is shown, and nucleotide position 37 is underlined. Adenosine peaks are indicated by a dashed line, guanosine peaks are bold. Because I prefers to base pair with C, I is represented as G.