Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek

The CDC33 (eIF4E) gene was amplified from S. cerevisiae genomic DNA using Pfu DNA polymerase (Fermentas) with eIF4Ef and eIF4Er primers containing NcoI and HindIII restriction sites as follows: 5 min at 95°C; then 25 cycles of 30 sec at 94°C, 30 sec at 55°C, and 2 min at 72°C; and finally, 10 min at 72°C. The PCR cassette was digested and inserted into the NcoI and HindIII restriction sites of pGEX-4T2 (GE Healthcare) to generate a pGEX4T2::CDC33 construct. A pYX212 yeast shuttle plasmid (Ingenius) was digested with EcoRI and XhoI restriction endonucleases. A sequence coding for K1 toxin was excised from a pYX213::M1 (Valis et al., 2006 ) plasmid using the same restriction enzymes and ligated into the digested pYX212 plasmid to generate the plasmid pYX212::M1. K2ORF3 was amplified from K. lactis pGKL2 plasmid DNA using Pfu DNA polymerase (Fermentas) with the NLS-ORF3 forward and the klorf3_2 reverse primers as follows: 5 min at 95°C; then 5 cycles of 30 sec at 94°C, 30 sec at 55°C, and 2 min at 72°C; and finally, 10 min at 72°C. One microliter of this reaction mixture was used for subsequent PCR amplification using Pfu DNA polymerase with ORF3_NLS_BamF and the klorf3_2 reverse primers as follows: 5 min at 95°C; then 20 cycles of 30 sec at 94°C, 30 sec at 55°C, and 2 min at 72°C; and finally, 10 min at 72°C. The resulting PCR product was purified using agarose electrophoresis and cloned into a pCR4-TOPO plasmid (Invitrogen). An NLS-ORF3 cassette from a pCR4-TOPO plasmid was excised with BamHI and SalI restriction endonucleases and inserted into the corresponding sites of the pYX212 or pYX213 (Ingenius) plasmids to generate pYX212::NLS-ORF3 or pYX213::NLS-ORF3 constructs, respectively. All clones were verified by restriction endonuclease digestion and sequencing. All primers used in this study are listed in Table S2 .