MCB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Long, M. B.
Right arrow Articles by Sullenger, B. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Long, M. B.
Right arrow Articles by Sullenger, B. A.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, October 1999, p. 6479-6487, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Evaluating Group I Intron Catalytic Efficiency in Mammalian Cells

Meredith B. Long and Bruce A. Sullenger*

Departments of Genetics and Surgery, Center for Genetic and Cellular Therapies, Duke University Medical Center, Durham, North Carolina 27710

Received 14 April 1999/Returned for modification 16 May 1999/Accepted 30 June 1999

Recent reports have demonstrated that the group I ribozyme from Tetrahymena thermophila can perform trans-splicing reactions to repair mutant RNAs. For therapeutic use, such ribozymes must function efficiently when transcribed from genes delivered to human cells, yet it is unclear how group I splicing reactions are influenced by intracellular expression of the ribozyme. Here we evaluate the self-splicing efficiency of group I introns from transcripts expressed by RNA polymerase II in human cells to directly measure ribozyme catalysis in a therapeutically relevant setting. Intron-containing expression cassettes were transfected into a human cell line, and RNA transcripts were analyzed for intron removal. The percentage of transcripts that underwent self-splicing ranged from 0 to 50%, depending on the construct being tested. Thus, self-splicing activity is supported in the mammalian cellular environment. However, we find that the extent of self-splicing is greatly influenced by sequences flanking the intron and presumably reflects differences in the intron's ability to fold into an active conformation inside the cell. In support of this hypothesis, we show that the ability of the intron to fold and self-splice from cellular transcripts in vitro correlates well with the catalytic efficiency observed from the same transcripts expressed inside cells. These results underscore the importance of evaluating the impact of sequence context on the activity of therapeutic group I ribozymes. The self-splicing system that we describe should facilitate these efforts as well as aid in efforts at enhancing in vivo ribozyme activity for various applications of RNA repair.

* Corresponding author. Mailing address: Box 2601 DUMC, Durham, NC 27710. Phone: (919) 684-6375. Fax: (919) 684-6492. E-mail: sulle001{at}mc.duke.edu.

Molecular and Cellular Biology, October 1999, p. 6479-6487, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals

Copyright © 1999 by the American Society for Microbiology. All rights reserved.