Escherichia coli strain
DH5α was used to amplify plasmids, and
E.coli transformations were performed using the high efficiency method of Inoue
et al. (13 (
link)). YPAD and synthetic complete medium (H-) were used as described previously (14 (
link)). Yeast strain
JD932 (
MATa ade2-1 trp1-1 ura3-1 leu2-3,112 his3-11,15 can1-100) (15 (
link)) was used for
in vivo measurement of programmed −1 ribosomal frameshifting. Yeast cells were transformed using the alkali cation method (16 (
link)). Dual luciferase plasmids pJD375 (
C1, no frameshift signal) and pJD376 (
F1, L-A virus
gag-pol frameshift signal) have been described previously (7 (
link)). Putative frameshift signals from
S.cerevisiae genes YOR026W/
BUB3 (
F2, plasmid pJD519) and YPL128C/
TBF1 (
F3, plasmid pJD478) were constructed as follows: (i) oligonucleotides from Integrated DNA Technology (Coralville, IA) were annealed and gel purified, and (ii) annealing the oligonucleotides 5′-TCGACAAAAAATCATCTTTCAGGGTGGATTGGAACGGCCCCAGTGATCCTGAGAACCCACAAAACTGGCCCG-3′ to 5′-GATCCGGGCCAGTTTTGTGGGTTCTCAGGATCACTGGGGCCGTTCCAATCCACCCTGAAAGATGATTTTTTG-3′ (
F2), and 5′-CGACAAATTTATCTCAAGCATCCTTCATCAGCTGCATCTGCTACTGAAGAGGG-3′ to 5′-GATCCTCTTCTGTAGCAGATGCAGCTGAAGAAGGATGCTGAGATAAATTTG-3′ (
F3) left overhanging single-stranded DNA complementary to SalI and BamHI restriction sites. The annealed oligonucleotides were ligated into p2mci (6 (
link)). The frameshift signal was sub-cloned as a SalI–EcoRI fragment into similarly digested pJD375. The open reading frame (ORF)
1a-1b frameshift signal from the SARS-associated Coronavirus (SARS-CoV) was cloned; sense 5′-GATCCTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTACTGATGTCGTCTACAGGGCTTTTGAGCT-3′ and antisense 5′-CAAAAGCCCTGTAGACGACATCAGTACTAGTGCCTGTGCCGCACGGTGTAAGACGGGCTGCACTTACACCGCAAACCCGTTTAAAAAG-3′ oligonucleotides were annealed, gel purified and cloned into BamHI and SacI restricted p2mc (6 (
link)). This was further sub-cloned into a pJD375-based plasmid where the reading frame was corrected using site-directed mutagenesis to add a cytosine downstream of the BamHI restriction site (
F4). A zero-frame control (
C2) plasmid was made by inserting two cytosine residues upstream of the BamHI restriction site and cells were grown in the absence or presence of 20 μg/ml of anisomycin (Sigma–Aldrich, St. Louis, MO). The annealed oligonucleotides were ligated into p2mci (6 (
link)). The SARS-CoV frameshift signal (
F4) was sub-cloned as a SalI–EcoRI fragment into similarly restricted pJD375.
In vivo DLAs for programmed −1 ribosomal frameshifting were performed in yeast strain JD1158 as described previously (7 (
link)). Luminescence readings were obtained using a Turner Designs TD20/20 luminometer (Sunnyvale, CA). Reactions were carried out using the Dual-Luciferase® Reporter Assay System from Promega Corporation (Madison, WI).
Jacobs J.L, & Dinman J.D. (2004). Systematic analysis of bicistronic reporter assay data. Nucleic Acids Research, 32(20), e160.
