FIG 1 .

Gaf1 positively regulates the transcription of isp7⁺. (A) Gaf1 is required for transcription of isp7⁺. Northern blot analysis of wild type (WT) or mutant cells carrying a deletion of the GATA transcription factor encoded by gaf1⁺, gaf2⁺, SPCC1393.08⁺, or ams2⁺ was carried out. Cells were grown to mid-log phase in rich medium YE (Yeast Extract) before RNA was extracted. rRNA was used as a loading control. (B) Gaf1 is required for the upregulation of isp7⁺ in response to a poor-quality nitrogen source. Wild-type or Δgaf1 cells were grown in minimal (M) medium and shifted to proline for 30 min (P). Total RNA was extracted and analyzed by RT-qPCR, using act1⁺ as a reference. Expression levels were quantitated using the comparative C_T method. (C) Gaf1 is physically associated with the promoter region of the isp7⁺ gene under nitrogen stress conditions. Gaf1 occupancy at the promoter region of isp7⁺ was assessed by ChIP analysis of chromatin extracts in Gaf1-HA strains. An untagged strain was used as control. Cells were grown in minimal (M) medium and shifted to proline for 1 h (P). Occupancy levels were quantified by real-time PCR. (D) Gaf1 is required for growth on proline. Serial dilutions of exponentially growing wild-type (WT), Δgaf1, Δgaf2, ΔSPCC1393.08, and Δams2 strains were spotted on YE medium, EMM, or proline medium.