BioProject

In filamentous strains of S. cerevisiae, nitrogen stress elicits a complex morphogenetic program resulting in the transition to a filamentous form of growth. More...

In filamentous strains of S. cerevisiae, nitrogen stress elicits a complex morphogenetic program resulting in the transition to a filamentous form of growth. The transcription factors Flo8p and Mss11p are both required for this filamentous growth transition, in that homozygous diploid flo8(delta/delta) and mss11(delta/delta) strains do not undergo filamentation under conditions of nitrogen stress. To identify genes that are regulated either directly or indirectly by the Flo8p and Mss11p transcription factors, we have implemented DNA microarray analysis to profile changes in mRNA levels in homozygous diploid strains of the filamentous (Sigma)1278b genetic background deleted for FLO8 and MSS11, respectively, under conditions of nitrogen stress. The transcriptional profiles will identify genes with altered transcriptional levels in the deletion mutants, serving as a means to identify cellular processes and signaling pathways regulated by Flo8p and Mss11p function. Overall design: Deletion mutants were constructed using a one-step polymerase chain reaction (PCR)-based gene disruption strategy with the G418 resistance cassette from plasmid pFA6a-KanMX6. After the haploid mutants were confirmed via PCR, the strains were allowed to mate on YPD+G418 plates for approximately 20 hours at 30 degrees Celsius. Mated cells were then streaked on SC-Trp-Leu plates to select for Y825 x HLY337 diploids. Yeast transformations were performed according to standard lithium acetate-mediated protocols with modifications. Yeast strains (wild-type and deletion mutants) were cultured under conditions of nitrogen stress (low ammonium media), and RNA was prepared using the Poly(A) Purist kit (Ambion, Austin, TX). RNA concentration and purity were determined spectrophotometrically and by gel electrophoresis. Microarray hybridization was performed with the Yeast Genome S98 Array using standard protocols (Affymetrix, Inc, Santa Clara, CA). Differentially expressed genes were identified by significance analysis of microarrays (SAM). SAM computed a nonparametric score for each gene by dividing the between-group difference of (normalized log) gene expression levels and the within-group difference of gene expression levels. The score was then compared with random permutation scores. The random permutation scores for a gene were computed in the same manner as the original score, but based on randomly sampled gene expressions. If the difference between the original score and the random permutation score was larger than a chosen threshold value, the corresponding change in gene expression was claimed to be significant. Each threshold value corresponded to a false discovery rate (the percentage of genes identified as being significant by chance alone). For all deletion comparisons in this study, we used SAM’s two-class unpaired analysis function, with significance thresholds selected so that the corresponding false discovery rate was 0. Less...