Author: Firth, Andrew E.; Wills, Norma M.; Gesteland, Raymond F.; Atkins, John F.
Title: Stimulation of stop codon readthrough: frequent presence of an extended 3' RNA structural element Document date: 2011_4_27
ID: 2u49b7xo_25
Snippet: In order to verify and investigate the functionality of the predicted RNA structures in VEEV and SINV, local sequences (15 nt 5 0 of the UGA stop codon and 156 nt 3 0 for VEEV or 204 nt 3 0 for SINV) were cloned in-frame between the renilla luciferase and firefly luciferase genes in vector pDluc. The firefly luciferase gene lacks an initiation codon and its expression is dependent on RT of the UGA codon. RT efficiencies were determined both in vi.....
Document: In order to verify and investigate the functionality of the predicted RNA structures in VEEV and SINV, local sequences (15 nt 5 0 of the UGA stop codon and 156 nt 3 0 for VEEV or 204 nt 3 0 for SINV) were cloned in-frame between the renilla luciferase and firefly luciferase genes in vector pDluc. The firefly luciferase gene lacks an initiation codon and its expression is dependent on RT of the UGA codon. RT efficiencies were determined both in vitro using rabbit reticulocyte lysate, and in HEK293 tissue culture cell lysates. A positive control for RT, the MuLV gag-pol RT site and 3 0 -adjacent pseudoknot, was included in all assays ( Figure 6A and B, lane 11).
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