To carry out microarray analyses, logarithmically growing wild type, Δgcn4 and Δyap1 mutant cells were split into two samples; one was treated with 20 mM boric acid for one hour while the other remained untreated. (A) Transporter genes that were upregulated or downregulated in response to boric acid treatment. (B) Amino acid biosynthesis genes that were upregulated and downregulated in response to boric acid treatment. Yeast Saccharomyces cerevisiae proteins annotation information from the yeast genome database (http://www.yeastgenome.org/ ) was used to identify transporters and other proteins. (C) Real time PCR analyses of ATR1 in wild type, Δgcn4 and Δyap1 cells in response to boron. Cells were treated or not with 50 mM boric acid for one hour and ATR1 expression was analyzed. Actin was used as the internal control. Bars represent the mean and SEM of triplicate assays.

(A) Real time PCR analysis of GCN4 mRNA in wild type cells treated with 50 mM boric acid for one hour. Actin was used as the internal control. (B) Wild type cells were transformed with the p180 plasmid which had the GCN4-lacZ reporter gene with all four uORFs of GCN4. Transformants were grown to logarithmic phase in YNB-URA medium. The cultures were split into two; one half was treated with 50 mM boric acid for one hour, and the other half remained untreated. β-galactosidase activities were measured as explained in . (C) Effect of boric acid treatment on Gcn4 protein levels determined by western blotting. Chromosomally tagged GCN4-MYC cells were grown to logarithmic phase, treated with indicated amounts of boric acid and harvested (900 µl, 0.5 OD₆₀₀). Primary (anti-c-myc) and secondary antibodies (anti-rabbit IgG) were used in a dilution of 1:3000 and 1:10000 respectively. The blot was stripped and reprobed yeast phosphoglycerate kinase monoclonal antibody as an internal control (1:1500 dilutions) and its 1:10000 diluted secondary antibody was goat anti mouse IgG conjugated with horseradish peroxidase.

(A) Overnight cultures of wild type and isogenic Δyap1, Δgcn4 yeast cells were first diluted to an OD₆₀₀ of 0.2, 0.02, 0.002 and 0.0002, then 5 µl of each cell suspension were spotted onto YPD plates which contained different amounts of boric acid, as indicated. The cells were grown for 3 days at 30°C and photographed. (B) ATR1 mRNA expression levels of wild type cells containing empty pAG423GPD vector, pAG423GPD-YAP1 and pAG423GPD-GCN4 vectors, respectively. Actin was used as an internal control. These experiments were performed three times. (C) Boric acid tolerance level of the same cells. The transformants were grown to logarithmic phase in YNB-HIS media, serially diluted to OD₆₀₀ of 0.2, 0.02, 0.002, and 0.0002 and 5 µl of cell suspension were spotted onto plates containing the indicated amounts of boric acid. The plates were incubated at 30°C for 5 days and photographed. (D) GCN4 mRNA expression levels in wild type cells overexpressing pAG423GPD and pAG423GPD-GCN4 vectors. (E) ATR1 mRNA levels in wild type and Δgcn4 mutant cells containing pAG423-YAP1 vector. Actin was used as an internal control.

(A) Radiogram (left panel) showing incorporation of radioactive methionine into newly synthesized proteins. Coomassie staining of the same gel is shown in the right panel as a loading control. (B) Boron treatment decreases the abundance of polysomes. Wild type cells were grown to logarithmic phase and then treated with 50 and 100 mM of boric acid or left untreated. The cell lysates were analyzed by sucrose density gradient centrifugation. The absorbance profiles at 254 nm show 40S, 60S, 80S and polysome fractions, as indicated.

(A) Wild type and Δgcn2 cells were grown to logarithmic phase in YPD medium; the cultures were split and treated with 25 mM or 50 mM boric acid for one hour or left untreated. The levels of eIF2α phosphorylation at Ser51 were measured by western blot analysis using an antibody against phospho Ser51 on eIF2α. The membrane was stripped and reprobed with antibodies against yeast phosphoglycerate kinase (Pgk) antibody as an internal control. (B) Effect of boric acid on eIF2α phosphorylation depends on uncharged tRNA binding to an intact HisRS-like domain in Gcn2. The Δgcn2 transformants carrying plasmids for an empty vector (p-only), GCN2 with S577A substitution (p-GCN2-S577A), GCN2 without HisRS domain (p-GCN2-m2), GCN2 without HisRS domain and with S577A substitution (p-GCN2-S577A-m2), and intact GCN2 (p-GCN2) were grown to logarithmic phase in YNB-URA medium and split in half; one of which was treated with 50 mM boric acid for one hour while the other was left untreated. Western blot analyses were performed with cell extracts using an antibody against eIF2α phosphorylated on Ser51. The membrane was stripped and reprobed with antibodies against yeast phosphoglycerate kinase (Pgk) as an internal control.