Fig. 3. Rgf3p is essential for cell viability and depletion of Rgf3p leads to a lysis phenotype similar to the depletion of Rho1p. (A) Genomic organization of the rgf3⁺ and rgf1⁺ loci, and deletion strategy for rgf3⁺ disruption. The direction of transcription is indicated by an arrow. Tetrads from a rgf3::ura4⁺/rgf3⁺ strain dissected on YES medium and incubated at 28°C for 4 days. (B) Terminal phenotype of rgf3-null mutants. Spores prepared from the rgf3::ura4⁺ strain were inoculated in MM lacking uracil and germinated for 18 hours. Cells were stained with rhodamine-conjugated phalloidin and DAPI to visualize F-actin and nuclei, respectively (top left) and with Calcofluor White (Cfw) to visualize the cell-wall material (top right). Spores with the wee1-50 rgf3 and sid2-250 rgf3 double mutations (prepared from strains YSM654 and YSM656, respectively) were inoculated in YES medium and germinated for 14 hours at 25°C and then for 6 hours at 36°C. Cells were stained with Hoechst and Cfw (bottom). (C) Lethal phenotype of the P81 nmt-rgf3 and P41 nmtrho1 shut-off mutants. Cells grown at 28°C in MM were supplemented with thiamine to repress the nmt promoter. Nomarsky micrographs were taken after 12 hours in MM with or without thiamine. (D) Growth phenotypes of P81 nmt-rgf3 and P41 nmt-rho1mutants under different growth conditions. Strains VT88 (81 nmt-rgf3⁺ + pREP81X) and PPG217 (rho1 + pREP41X nmt-rho1⁺) were streaked onto several plate media (YES, YES + Sorbitol and MM-leu) and the plates were incubated for 3 days at 28°C. The nmt promoter is off in rich medium (YES) and on in MM. Strain VT88 carried pREP81X, an empty plasmid, to allow cells to grow in MM-leu.