Abstract:
:A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell types.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Ilegems E,Pick HM,Vogel Hdoi
10.1093/nar/gnf128keywords:
subject
Has Abstractpub_date
2002-12-01 00:00:00pages
e128issue
23eissn
0305-1048issn
1362-4962journal_volume
30pub_type
杂志文章abstract::An updated compilation of 300 E. coli mRNA promoter sequences is presented. For each sequence the most recent relevant paper was checked, to verify the location of the transcriptional start position as identified experimentally. We comment on the reliability of the sequence databanks and analyze the conservation of kn...
journal_title:Nucleic acids research
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journal_title:Nucleic acids research
pub_type: 杂志文章
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pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:1987-11-25 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2013-01-07 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl396
更新日期:2006-06-06 00:00:00
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journal_title:Nucleic acids research
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更新日期:2009-07-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2014-09-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkp097
更新日期:2009-04-01 00:00:00
abstract::PIECE (Plant Intron Exon Comparison and Evolution) is a web-accessible database that houses intron and exon information of plant genes. PIECE serves as a resource for biologists interested in comparing intron-exon organization and provides valuable insights into the evolution of gene structure in plant genomes. Recent...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw935
更新日期:2017-01-04 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/11.19.6611
更新日期:1983-10-11 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.19.4556
更新日期:1993-09-25 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw432
更新日期:2016-07-27 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/2.11.2077
更新日期:1975-11-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/17.23.9679
更新日期:1989-12-11 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:2005-03-22 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2003-01-15 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2007-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
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更新日期:2020-05-07 00:00:00
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
pub_type: 杂志文章
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