Split-Doa10: A Naturally Split Polytopic Eukaryotic Membrane Protein Generated by Fission of a Nuclear Gene
Figure 4
A transcriptional promoter in the KlDOA10 IVS.
A. Schematic representation of the KlDOA10 gene consisting of the Nt-ORF, the IVS and the Ct-ORF. The annealing sites of the Nt and Ct ORF-specific hybridization probes are depicted as black horizontal bars. B. Northern blot analysis of KlDOA10 transcripts with a KlDOA10 Nt-ORF specific probe (Nt-probe). Total RNA was analyzed from WT and doa10Δ K. lactis cells. A picture of the ethidium bromid-stained agarose gel before transfer to membrane is shown on the left (gel). The PhosphorImager-scanned image on the right was derived from a 3 d exposure of the Nt probe-hybridized membrane. M, RNA size markers (in nts). The horizontal bar on the right marks the position of the ∼1200-nt KlDOA10 Nt-ORF-specific transcript. C. Northern blot analysis of KlDOA10 transcripts with a KlDOA10 Ct-ORF specific probe (Ct-probe). Northern blotting was done as in Fig. 4B except a [32P]-labeled Ct-probe was used for hybridization. The hybridization image is from a 7 d film exposure (Ct-probe). The horizontal bar marks the position of the ∼3200-nt KlDOA10 Ct-ORF-specific transcript. D. IVS promoter activity in the absence of other KlDOA10 sequences. Left: Schematic representations of the IVS-URA3-HA and the ScGPD-URA3-HA sequences that were inserted into a promoterless K. lactis low-copy plasmid. The constructs were transformed into K. lactis cells, and expression of the Ura3-HA reporter was examined by anti-HA immunoblotting (right). A nonspecific background band (bkg) served as a loading control. E. Mapping of the 5′ border of the promoter contained within the IVS. Left: Schematic representation of the IVS-Ct-ORF-13MYC sequence and 5′-truncated versions of the IVS that were cloned into a promoterless K. lactis low-copy plasmid. The different constructs were transformed into K. lactis cells and expression of the Ct-13MYC fragment was examined by anti-MYC immunoblotting (right). A nonspecific background band (bkg) served as a loading control.