Figure 5.

The inositol auxotrophy of Δsac1 strains is not the consequence of defects in INO1 expression. (A) The yeast strains with the indicated genotypes were streaked for isolation on uracil-deficient medium in the presence (+Inositol) or absence (−Inositol) of inositol and incubated at 26°C for 72 h. The YEp(P_SEC14:: INO1) plasmid, which drives constitutive expression of INO1 from the SEC14 promoter, was incapable of rescuing inositol prototrophy in the Δsac1 strain CTY244. That this plasmid effectively drove INO1 expression is amply demonstrated by its ability to rescue growth of the ino1-13 mutant (CTY) on inositol-free medium. (B) Wild-type (CTY182) and Δsac1 (CTY244) strains were grown in YPD medium to midlogarithmic growth phase, harvested, and washed. The cultures were split and either shifted to minimal medium containing both inositol and choline (+Ino) or to inositol- and choline-free medium (−Ino). After 4 h of incubation at 30°C with shaking, cells were harvested, and total RNA was recovered and subjected to Northern analysis using specific probes for INO1 and OPI3 mRNA that were generated by random priming. Each lane was loaded with 10 μg of RNA. The results clearly demonstrate that both INO1 and OPI3 expression was induced in cells shifted to inositol- and choline-free medium. To confirm proper normalization of RNA load, these same RNA samples were also probed for messages (e.g., PSS1) that are present under both the Ino⁻ and Ino⁺ conditions. As expected, PSS1 mRNA was detected in all lanes (our unpublished results).