Expression in vivo of a mutant human initiator tRNA gene in mammalian cells using a simian virus 40 vector.

ABSTRACT

We have cloned both the wild type (A54) and mutant (T54) human initiator genes described in the preceding paper (Drabkin, H. J., and RajBhandary, U. L. (1985) J. Biol. Chem. 260, 5580-5587) as 141-base pair fragments into the SV40-pBR322 vector pSV1GT3. These vectors were subsequently used to transfect monkey kidney CV-1 cells to obtain recombinant virus stocks carrying each of the initiator tRNA genes. Following infection of CV-1 cells by the recombinant virus stocks, both the wild type and mutant tRNAs are produced in large quantities during a 48-h period. Fingerprint analysis of 32P-labeled tRNAs was used to characterize the tRNAs made in vivo and to show that the sequence AUCG in loop IV of the wild type tRNA is replaced by T psi CG in the mutant tRNA. Modified nucleotide composition analysis of the [32P]tRNAs overproduced in vivo shows that they contain all the modified nucleotides found in human placenta initiator tRNA. Both wild type and mutant initiator tRNAs can be aminoacylated by either mammalian or Escherichia coli methionyl-tRNA synthetases. Furthermore, the mutant tRNA can be easily separated from the endogenous monkey initiator tRNA by RPC-5 column chromatography.