Nucleic Acids Research

A combination of several enzymes, RNase-T.. , nuclease S-, T.-polynucleotide kinaee and T.-RNA ligase were used -to prepare and modify different fragments of feast tRNAP (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 O 2 P ) labelled yeast tRNA Sp were raicroinjected 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 veast tRNAu? were quite stable inside the frog oocyte. Also, the G^ **was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q^; U ^ into mcm5s^U and mcm5u. In contrast, C ^ and A34 were not transformed at all neither in the cytoplasm nor in tne 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.