Figure 5. Deletion of gaf1⁺ results in accelerated induction of ste11⁺ expression under nitrogen-starved conditions.

(A) Northern blot analysis of ste11⁺ and gaf1⁺ mRNA from wild-type and gaf1Δ cells exposed to nitrogen starvation. Cells of wild-type (972) and gaf1Δ (KL230) strains pre-grown in EMM2 (+N) were shifted to EMM-N (−N) and cultured with constant shaking. At indicated time points, cells were harvested and washed twice with distilled water, and total RNAs were extracted from the cells. RNA blots were hybridized with ³²P-labeled PCR-amplified gaf1⁺ and ste11⁺ probes. For internal control, all blots were stripped and subsequently rehybridized with ³²P-labeled actin-specific probe (act1⁺). (B) β-Galactosidase reporter assay for analysis of ste11⁺ expression in wild-type and gaf1Δ cells subjected to nitrogen starvation. Cells of wild-type (ED665) and gaf1Δ (KL240) strains carrying pJLC-Ste11_(p)-LacZ were cultivated to mid-log phase in EMM2 (+N) and shifted to EMM-N (−N). At indicated time points, cells were harvested and washed twice with distilled water, and the level of ste11 ⁺ expression was estimated by measuring the activity of β-galactosidase in each sample. Values are the mean ± standard error of three independent experiments carried out in triplicate, n = 9. *, p<0.01; **, p<0.05 (two-tailed Student's t-test, versus wild-type). WT denotes the wild-type (gaf1⁺).