Figure 3.

Transcriptional and translational downregulation of HO expression in response to mating pheromone. (a) Relative levels of HO mRNA (normalized to 1.0 for the long transcript at time = 0 min) as a function of time after α-factor treatment. RNA was prepared and analyzed as in Figure 2c. Closed circles show values for both forms of transcripts and open circles represent values for the long HO transcript. (b) Northern analysis of total RNA (10 µg) from growing cells (lane 1) or cells treated with α-factor for 30 minute (lane 2). The blot was stripped and re-probed for ACT1 as a loading control. (c) Relative levels of HO mRNA across polysome gradients in growing cells (filled squares) or cells treated with α-factor for 30 minute (open circles). Gradients were performed and analyzed as described in Figure 2 using PCR primers recognizing both HO transcripts. The top of the gradient is to the left and the position of the 80S monosome is marked with the arrow. (d) Relative levels of the long forms of HO across polysome gradients in growing cells (filled circles, right axis) or cells treated with α-factor (open circles, left axis). QPCR using primers specific to the long forms of HO was performed on cDNA obtained from the same RNA samples used in the experiment described in panel c of this figure. Note the difference in scale on the two axes.