Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements Arun Venkatesan 1 and Asim Dasgupta 1 , 2 , * Molecular Biology Institute, University of California, 1 and Department of Microbiology, Immunology and Molecular Genetics, UCLA School of Medicine, 2 Los Angeles, California 90095 ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular and Cellular Biology American Society For Microbiology

Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

Molecular and Cellular Biology , Volume 21 (8): 2826 – Apr 15, 2001

Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

Molecular and Cellular Biology , Volume 21 (8): 2826 – Apr 15, 2001

Abstract

Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements Arun Venkatesan 1 and Asim Dasgupta 1 , 2 , * Molecular Biology Institute, University of California, 1 and Department of Microbiology, Immunology and Molecular Genetics, UCLA School of Medicine, 2 Los Angeles, California 90095 ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

Loading next page...
 
/lp/american-society-for-microbiology/novel-fluorescence-based-screen-to-identify-small-synthetic-internal-PnZQYeaX0Q

References (48)

Publisher
American Society For Microbiology
Copyright
Copyright © 2001 by the American society for Microbiology.
ISSN
0270-7306
eISSN
1098-5549
DOI
10.1128/MCB.21.8.2826-2837.2001
pmid
11283261
Publisher site
See Article on Publisher Site

Abstract

Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements Arun Venkatesan 1 and Asim Dasgupta 1 , 2 , * Molecular Biology Institute, University of California, 1 and Department of Microbiology, Immunology and Molecular Genetics, UCLA School of Medicine, 2 Los Angeles, California 90095 ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

Journal

Molecular and Cellular BiologyAmerican Society For Microbiology

Published: Apr 15, 2001

There are no references for this article.