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Mol Cell Biol, August 1998, p. 4418-4425, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

Ho-Jin Park and Uttam L. RajBhandary*

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 13 March 1998/Returned for modification 30 April 1998/Accepted 11 May 1998

As an approach to inducible suppression of nonsense mutations in mammalian cells, we described recently an amber suppression system in mammalian cells dependent on coexpression of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) along with the E. coli glutamine-inserting amber suppressor tRNA. Here, we report on tetracycline-regulated expression of the E. coli GlnRS gene and, thereby, tetracycline-regulated suppression of amber codons in mammalian HeLa and COS-1 cells. The E. coli GlnRS coding sequence attached to a minimal mammalian cell promoter was placed downstream of seven tandem tetracycline operator sequences. Cotransfection of HeLa cell lines expressing a tetracycline transactivator protein, carrying a tetracycline repressor domain linked to part of a herpesvirus VP16 activation domain, with the E. coli GlnRS gene and the E. coli glutamine-inserting amber suppressor tRNA gene resulted in suppression of the amber codon in a reporter chloramphenicol acetyltransferase gene. The tetracycline transactivator-mediated expression of E. coli GlnRS was essentially completely blocked in HeLa or COS-1 cells grown in the presence of tetracycline. Concomitantly, both aminoacylation of the suppressor tRNA and suppression of the amber codon were reduced significantly in the presence of tetracycline.

* Corresponding author. Mailing address: Rm. 68-671A, MIT, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-4702. Fax: (617) 252-1556. E-mail: bhandary{at}wccf.mit.edu.

Mol Cell Biol, August 1998, p. 4418-4425, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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