A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C). This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position. Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions. Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte. Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U. In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte. The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.

We have investigated the specificity of the enzymes Q‐insertase and mannosyl‐Q transferase that replace the guanosine at position 34 (wobble base) in the anticodon of several tRNAs by Q or mannosyl‐Q derivatives. We have restructured in vitro the normal anticodon of yeast tRNA‐Asp‐GUC, yeast tRNAArgICG and yeast tRNALeuUAG. With yeast tRNA‐Asp‐GUC, we have replaced one or several nucleotides in the vicinity of G34 by one of the four canonical nucleotides or by pseudouridylic acid; we have also constructed a tRNAAsp with eight bases instead of seven in the anticodon loop. With yeast tRNAArgICG and yeast tRNALeuUAG, we have replaced their anticodon by the trinucleotide GUC, coding for aspartic acid. The chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and after 72 h the amount of Q34 and mannosyl‐Q34 incorporated was measured. Our results show that the U33G34U35 sequence, within an anticodon loop of seven bases in chimerical yeast tRNA‐Asp‐GUC, tRNAArgGUC or tRNALeuGUC, is the main determinant for Q‐insertase activity at position 34; the rest of the tRNA sequence has only a slight influence. For mannosyl‐Q transferase, however, a much broader structural feature of the tRNA than just the U33G34U35 sequence is important for the efficiency of Q34 transformation into mannosyl‐Q34.