Fig. 3. The HO 3′-UTR is sufficient for MPT5-mediated repression of HO. (A) Effect of MPT5 overexpression on ADE2-3HA reporter strains. Yeast strains TTC59 (ADE2-3HA-HO 3′-UTR, GAL1p-MPT5) and TTC62 (ADE2-3HA-ADH1 3′-UTR, GAL1p-MPT5) were streaked on the SC (glucose)-Ade or SG (galactose)-Ade plates and incubated for 3 days at 30°C. (B) Effect of MPT5 overexpression on Ade2-3HA protein levels. Yeast cells were cultured in 2% raffinose medium at 30°C and treated with galactose (2%) to induce MPT5 expression from GAL1p-MPT5. At the times indicated, cells were harvested and western blot analysis was performed to assay the level of Ade2-3HA protein (top). Tubulin protein (bottom) was included as a quantity control. (C) Effect of MPT5 overexpression on ADE2-3HA mRNA levels. Yeast cells were harvested as in (B), and northern blot analysis was performed to assay the level of ADE2-3HA mRNA (top). ACT1 mRNA (bottom) was included as a quantity control. (D) Effect of MPT5 overexpression on degradation of mRNA. Yeast strain TTC181 (GAL1p-ADE2-3HA-HO 3′-UTR) harboring YEp195-MPT5 or empty vector was incubated in SR-Ura. Transcription was induced by adding galactose to a final concentration of 2%. The culture was then transferred to medium containing 2% glucose. Aliquots were removed at various times, and the amounts of the ADE2 transcript were quantitated by dot blotting as described in Materials and methods. Decay rates were determined from semilog plots of the percentage of hybridizing material remaining at different times after the inhibition of transcription.