Therefore, it appears that Δphx1/Δphx1 diploid cells are FGFR inhibitor defective in this website completing the first meiotic division . The sporulation efficiency was determined by counting the number of asci among at least 500 cells counted. Compared with the wild-type cells which demonstrated up to about 50% sporulation efficiency, the mutant diploids exhibited only about 10% efficiency (Figure 6B). Figure 6 Sporulation defect of Δphx1/Δphx1 mutant diploid. (A) The wild type and mutant diploid cells were grown to the stationary phase (OD600 of 8–9; ~70 h culture) in EMM at 30°C and examined
under the microscope (Axiovert 200 M, Carl Zeiss). Representative DIC and DAPI images were presented. (B) Quantification of the sporulation efficiency. Diploid
cells grown for different lengths of time at 30°C in EMM were examined under the microscope to count the number of spore-containing asci. The percentage of asci formation among a total of more than 500 counted cells was presented as sporulation efficiency. Cells grown from three independent cultures were examined 3-Methyladenine in vivo to obtain average values. Conclusions Phx1 is a homeobox-containing protein whose synthesis is elevated during the stationary phase. It resides primarily in the nucleus and contains the transcriptional activating ability when bound to DNA, supporting its role as a transcriptional regulator. Its synthesis is induced by nutrient starvation, various oxidative stresses, and by heat shock, coinciding with its role in long-term survival and stress resistance. It is also critically required for the formation of meiotic spores from diploid cells. Taken all these observations together, it is quite clear that Phx1 is a novel regulator that confers cells with fitness to survive during the nutrient-lacking stationary phase. Amino acid It enhances viability and ability to form spores for the future, most likely through reprogramming gene expression pattern. Elucidation of the signaling pathway as well as its target genes will be of interest to understand the mechanism of long-term survival and sporulation specific in this fungi as well
as common across other organisms. Methods Strains, plasmids and culture media We used ED665 (h − ade6-M210 leu1 32 ura4 D18), ED668 (h + ade6 M216 leu1 32 ura4 D18), JH43 (h − ade6 M210 leu1 32) and 972 (h – ) strains as the wild type . To disrupt the phx1 + gene, we replaced 2200 nt of the phx1 + ORF in pUC18-phx1 + recombinant plasmid with a ura4 + cassette . Digestion of pUC18-Δphx1::ura4 + with ClaI/BglII generated a 4.3 kb fragment, which was used to transform wild-type cells to create mutant strains ESX5 (Δphx1::ura4 + in ED665) and ESX8 (Δphx1::ura4 + in ED668). Transformants were confirmed by both Southern hybridization and PCR. We also generated the prototrophic Δphx1 mutant without auxotrophic markers.