Figure 7.

Non-essential amino acids isoleucine, threonine, and valine suppress GCN4 expression. The WT strain was transformed with plasmids p164 (GCN4) or p238 (GCN4^C, which constitutively expresses Gcn4p) and CLS was measured in synthetic dextrose minimal medium without (SD) or with (SD + ITV) the non-essential amino acids isoleucine, threonine, and valine added to final concentrations listed for SC1 (Table 2). A. Viability is expressed in colony forming units (CFU) per ml of culture and is plotted as the log of the percent of viability on day 1. B. Cell density (OD₆₀₀) was measured on days 0, 1, 3, 5, and 8. C. Measurement of Gcn4p levels was done during chronological aging. WT cells transformed with p164 (GCN4) or p238 (GCN4^C) and grown in SD or SD + ITV were harvested on days 0, 1, or 3. Equal amounts of whole cell lysates (based on OD₆₀₀ units) were analyzed by Western blotting with an affinity-purified antibody to Gcn4p (see Materials and Methods). The intensities of Gcn4p bands (arrowhead) were quantified using ImageJ software and normalized to the intensities of bands (asterisk) detected non-specifically in a gcn4Δ strain (Δ). Values for the normalized Gcn4p band intensities relative to lane 8 are shown below each lane (Gcn4p). Positions of molecular weight markers are shown on the left (in kDa).