Cellular debris was removed by centrifugation at 12,000 g for 30 minutes at 4 C. The supernatants were incubated with anti GFP antibodies overnight at 4 C. After incubation, protein G Sepharose was used for precipitation. The beads were washed with TSPI buffer four times and then eluted with SDS sample buf fer for immunoblot analysis. selleck chemicals Pazopanib Statistical analysis Densitometric analysis of immunoblots from three inde pendent experiments was performed using ImageJ windows version. The data were analyzed using windows version of Origin 6. 0 or Prism 5. The pictures in Figure 1A were draw using DOG 1. 0. Background TRA1 is an essential gene in Saccharomyces cerevisiae that encodes a 437 kDa protein product.
Inhibitors,Modulators,Libraries It is a member of a family of key signaling and regulatory Inhibitors,Modulators,Libraries molecules that con tain a C terminal phosphatidylinositol 3 kinase domain and is a Inhibitors,Modulators,Libraries component of two multisubunit tran scriptional regulatory complexes, the SAGA SLIK and NuA4 complexes, which also contain the histone acetyl transferase enzymes, Gcn5 and Esa1, respectively. Tra1 interacts directly with transcriptional activator pro teins and is thought to be critical in recruitment of SAGA SLIK and NuA4 to their target promoters. Previously we identified mutations in the C terminal PI3K domain of Tra1 that showed defects in transcriptional activation, sensitivity to ethanol and the cell wall destabi lizing agent calcofluor white and resulted in shortened tel omeres. The pattern of changes neither fully mimicked those seen upon disruption of other SAGA SLIK nor NuA4 components.
For example, unlike strains with deletions of NuA4 components, the tra1 mutant Inhibitors,Modulators,Libraries strains were relatively insensitive to DNA damaging agents. We performed an initial systematic genetic array analysis with the most pronounced allele, tra1SRR3413. This analysis did not identify any synthetic lethal Inhibitors,Modulators,Libraries interactions but did reveal 23 synthetic slow growth interactions, many in combination with deletions of genes involved in cell membrane wall processes. As the lack of synthetic lethal interactions may have arisen from an incomplete selection against diploids in the automated screens, we repeated the screen in a strain background that selects more strongly against dip loids. This analysis identified 114 genes displaying syn thetic sick lethal interactions with tra1SRR3413.
Genes involved in transcription, RNA processing, selleck chem inhibitor mitochondrial function and membrane sorting protein trafficking were prevalent. The phenotypes and genetic interactions of these strains also point to a role for Tra1 in the cellular response to stress. Results SGA analysis of tra1SRR3413 As a component of both SAGA and NuA4 complexes, Tra1 is positioned to play a major role in nuclear processes. Previously we performed an SGA analysis on Tra1 using an allele that partially impairs function. No synthetic lethal interactions were obtained in this analysis.