Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene.
As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells. The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence. This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells. We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor. Concomitant expression of the E. coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor. These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E. coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner.
Drabkin
H J
HJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Park
H J
HJ
RajBhandary
U L
UL
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D018389:Codon, Nonsense / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D000882:Haplorhini; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*
D018389:Codon, Nonsense; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases