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

(41). For 5' RACE, RT-PCR was carried out as follows: 0.5 μg of total yeast RNA and 0.15 μg of random primers (Invitrogen) were used for cDNA synthesis using 100 U of SSC III Reverse Transcriptase (Invitrogen) in a 20 μl reaction (50°C for 99 min). After the cDNA purification using the High Pure PCR Product Purification Kit (Roche), 800 U of recombinant TdT (Roche) and 0.5 mM dGTP (Fermentas) were used for cDNA tailing in 1x TdT buffer without CoCl 2 for 30 min at 37°C with subsequent TdT inactivation at 70°C for 10 min. For amplification of cDNA, 2.5 μl of the reaction mixture was used for the following PCR with the universal olig2(dC) anchor primer and an appropriate gene-specific primer. In the case of 3' RACE, 0.5 μg of total yeast RNA was polycytidinylated using a Poly(A) Tailing Kit (Applied Biosystems) with 2 mM CTP in a total volume of 25 μl for 90 min. Reverse transcription was then performed using oligo(dG)anch2 primer as indicated above. After cDNA purification using the High Pure PCR Product Purification Kit (Roche), 2.5 μl of the purified cDNA was used for a subsequent PCR reaction with the universal primer anch2 and an appropriate gene-specific primer. In both types of RACE experiments, following PCR amplification and electrophoresis, the corresponding fragments were purified from gel using a FastBack DNA minispin kit (Renogen Biolab), cloned into a pCR4-TOPO plasmid using the TOPO strategy and sequenced using the universal T7 promoter primer and/or a T3 primer. All primers used in this study are listed in Table S2 .