IRES activity identification of 5’UTRs of KOG1 and GCN2 mRNA detected by RNA-Seq. (A) Construction of vectors. The bicistronic vector pRml-LacZ containing the RML gene and the LacZ gene used as the negative control. Candidate IRESes were inserted between the RML gene and the LacZ gene, generating the pRml-UTR-LacZ. The expression of LacZ ORF in another control plasmid pLacZ was controlled by the GAP promoter. The arrows represent the primers used in PCR identification. (B) Detection of RML and β-galactosidase on MDT plate and MDX plate, respectively. (C) Identification of the integrity of bicistronic mRNAs with primer P1/P2. (D) Northern blot analysis of the transcripts from total RNA isolated from P. pastoris X33/pRml-LacZ, X33/pRml-GCN2-LacZ, X33/pRml-KOG1-LacZ, and X33/pLacZ.
Liang et al. BMC Genomics 2012 13:738 doi:10.1186/1471-2164-13-738