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Molecular and Cellular Biology, May 2000, p. 3116-3124, Vol. 20, No. 9
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Suppression of Nonsense Mutations in Cell Culture and Mice by Multimerized Suppressor tRNA Genes

Massimo Buvoli, Ada Buvoli, and Leslie A. Leinwand*

Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309

Received 22 November 1999/Returned for modification 5 January 2000/Accepted 8 February 2000

We demonstrate here the first experimental suppression of a premature termination codon in vivo by using an ochre suppressor tRNA acting in an intact mouse. Multicopy tRNA expression plasmids were directly injected into skeletal muscle and into the hearts of transgenic mice carrying a reporter gene with an ochre mutation. A strategy for modulation of suppressor efficiency, applicable to diverse systems and based on tandem multimerization of the tRNA gene, is developed. The product of suppression (chloramphenicol acetyltransferase) accumulates linearly with increases in suppressor tRNA concentration to the point where the ochre-suppressing tRNASer is in four- to fivefold excess over the endogenous tRNASer. The subsequent suppressor activity plateau seems to be attributable to accumulation of unmodified tRNAs. These results define many salient variables for suppression in vivo, for example, for tRNA suppression employed as gene therapy for nonsense defects.

* Corresponding author. Mailing address: Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Campus Box 347, Boulder, CO 80309-0347. Phone: (303) 492-7606. Fax: (303) 492-8907. E-mail: Leslie.Leinwand{at}Colorado.edu.

Molecular and Cellular Biology, May 2000, p. 3116-3124, Vol. 20, No. 9
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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