Figure 3

Marked Pch PRP8 inteins splice and allow genetic selection in yeast. The inteins described in Figure 2, but with different exteins, were expressed in yeast under the control of the GAL1 promoter. For expression in yeast, the N-extein was GFP with a nuclear localization signal, and the C-extein was a FLAG epitope tag. The spliced extein product is NLS-GFP-FLAG (33 kD). The molecular weights of the Pch marker-inteins are given in Figure 2. However in the anti-GFP and anti-FLAG Westerns there is no clear evidence for unspliced or partially spliced products of higher molecular weight than the spliced product, such as seen in Figure 2. A. Western blots showing the high efficiency of splicing in strain YL (2,4) LU expressing the modified inteins (and controls) from the following plasmids: Lane 1, pBM258; Lane 2, pRR26; Lane 3, pRR21; Lane 4, pRR22; Lane 5, pRR23; Lane 6, pRR24; Lane 7, pRR25. Analysis was performed as described in Methods. The genetic markers of the inteins and antibodies used for Western blots are indicated above and below the gels, respectively. Note that in the anti-FLAG western a background band was apparent in all lanes, including the protein extracts from the vector alone and NLS-GFP expression in lanes 1 and 2. B. Five-fold serial dilutions of the various yeast strains were prepared and spotted onto SGal-ura medium, SGal-ura medium lacking histidine or leucine, or SGal-ura supplemented with antibiotic as indicated. The plasmid backbone confers uracil prototrophy.