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

H J Drabkin,U L Rajbhandary

doi:10.1016/s0021-9258(18)89063-9

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.

Highlights

We have cloned both the wild type (A54) and mutant binant vectors carrying thewild type or mutant tRNA genes (T54)human initiator genes describedin the preceding paper
We show that is the mutant tRNA overproduced in vivo and that itcanbeaminoacylatedwithmethionineand separated from the endogenous CV-1 initiator tRNA
AUCG in loop IV of the wild typetRNA is replaced by quantities of the mutant initiator tRNfAor i n vitro functional

Summary

RESULTS

We have reported(DrabkinandRajposition analysis of the [“PItRNAs overproduced in Bhandary, 1985b) on attempts to achieve expression of the vivo shows that they contain all the modified nucleo- human initiator tRNA gene in yeast. Analysis of viral DNA obtained from Hirt extractsof cells infected with the various virus stocks by HindIII digestion revealed that the wild type (SVA54) clone chosen for study contained three tandemcopies of the initiator tRNA gene (Fig. 2). This is clearly seen in the Southernhybridiza-. Other analyses (data not shown) usiandgifferent solvent system demonstrate the presenceof this nucleotide

The Mutant tRNA Is More Readily Denatured than the

NATIVE d*

DISCUSSION

Haferlals and Methods