versicolor root tips with haustoria induced by 20 um DMBQ. Alignment of the 5, 6, and 4 independ ently recovered 50 RACE, 30 RACE, and full length cDNA clones, respectively, revealed a TvPirin open read ing frame encoding a 288 amino acid long small molecule protein with a 50 untranslated region of 64 68 base pairs. However, the previ ously published TvPirin cDNA sequence has a coding cap acity for a 322 aa long protein. This cDNA se quence was created by contig assemblies Inhibitors,Modulators,Libraries following 454 and Illumina sequencing of the T. versicolor transcrip tome. In comparison to the sequence of the TvPirin full length cDNA reported here, that of the TrVeBC2 244713 clone contains an additional 80 bp in the 50UTR and an extra 100 bp in the 30UTR.
The extended 50UTR in clone TrVeBC2 244713 contains an ATG codon that was assigned as the translation Inhibitors,Modulators,Libraries start codon and explains the longer coding sequence of the previously published TvPirin ORF. In order to resolve these discrepancies regarding the TvPirin transcript 50UTR and ORF, we performed in silico experiments and quantitative transcriptional assays. First, BLAST searches for the presence of other TvPirin contig assemblies from the T. versicolor transcriptome uncovered seven clones with homology to the TvPirin cDNA clone TrVeBC2 244713. Two of these clones had the same orientation as the TvPirin cDNA and lacked the extended 50UTR. The remaining 5 clones had the reverse orientation and 50 UTRs of variable lengths. One of these clones had the same length and shared 100% identity in reverse complementation order to the TrVeBC2 244713 TvPirin cDNA.
Another reverse complemented clone shared discon tinuous identity with the TvPirin cDNA sequences in both orientations. Inhibitors,Modulators,Libraries These observations suggest that these seven clones represent either transcripts from genes other than Inhibitors,Modulators,Libraries TvPirin or artifacts of the contig assembly. Similar BLAST searches in the T. versicolor SSH root tip contig libraries enriched for transcripts up regulated by DMBQ identified two clones, Contig5118 and Contig492, with homologies to the 50 and 30 half of our TvPirin cDNA se quence, respectively, and without the extended 50UTR of the TrVeBC2 244713 clone. Second, we performed RT qPCR on total RNA isolated from T. versicolor root tips following exposure to Inhibitors,Modulators,Libraries two HIFs and a non HIF, using primers chosen for amplification of either the extended 50UTR or the shorter 50UTR of the TvPirin transcripts. The shorter 50UTR amplicon displayed the same up regulated expression pattern contain as the TvPirin ORF amplicon, whereas the prolonged 50UTR amplicon did not show any significant up regulation in response to either DMBQ or peonidin, indicating that the extended 50UTR does not authentically belong to the TvPirin transcript.