We have determined the nucleotide sequence of the major species of E. coli tRNASer and of a minor species having the same GGA anticodon. These two tRNAs should recognize the UCC and UCU codons, the most widely used codons for serine in the highly expressed genes of E. coli. The two sequences differ in only one position of the D-loop. Neither tRNA has a modified adenosine in the position 3'-adjacent to the anticodon. This can be rationalized on the basis of a structural constraint in the anticodon stem and may be related to optimization of the codon-anticodon interaction. Comparison of all E.coli serine tRNAs (and that encoded by bacteriophage T4) reveals characteristic (possibly functional) features. Evolutionary analysis suggests an eubacterial origin of the T4 tRNASer gene and the existence of a recent common ancestor for the tRNASerGGA and tRNASerGUC genes.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.

We have investigated the specificity of the enzyme tRNA (wobble guanosine 2′‐O‐)methyltransferase which catalyses the maturation of guanosine‐34 of eukaryotic tRNAPhe to the 2′‐O‐methyl derivative Gm‐34. This study was done by micro‐injection into Xenopus laevis oocytes of restructured yeast tRNAPhe in which the anticodon GmAA and the 3′ adjacent nucleotide ‘Y’ were substituted by various tetranucleotides. The results indicate that the enzyme is cytoplasmic; the chemical nature of the bases of the anticodon and its 3′ adjacent nucleotide is not critical for the methylation of G‐34; the size of the anticodon loop is however important; structural features beyond the anticodon loop are involved in the specific recognition of the tRNA by the enzyme since Escherichia coli tRNAPhe and four chimeric yeast tRNAs carrying the GAA anticodon are not substrates; unexpectedly, the 2′‐O‐methylation is not restricted to G‐34 since C‐34, U‐34 and A‐34 in restructured yeast tRNAPhe also became methylated. It seems probable that the tRNA (wobble guanosine 2′‐O‐)methyltransferase is not specific for the type of nucleotide‐34 in eukaryotic tRNAPhe; however the existence in the oocyte of several methylation enzymes specific for each nucleotide‐34 has not yet been ruled out.

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.

An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.