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Differentiation of stationary phase Saccharomyces cerevisiae cultures
Results of the Viabilty and ROS assays
Achievement/Results
Survival of all organisms requires the ability to withstand periods of starvation. A model for studying these responses is the entry of cultures of the yeast Saccharomyces cerevisiae into stationary phase (SP) as a result of carbon starvation. Important features of cells in SP that suggest this process is analogous to important differentiation processes in other organisms. Yeast SP yeast cultures are composed of two distinct cell fractions, quiescent (Q) and non-quiescent (NQ), which are separable by density. We have examined this process by transcriptome & proteome analysis & are working on using pools of more than 7000 yeast strains from 3 different yeast strain sets, to identify & characterize genes required for entry into SP & the formation of Q & NQ cell fractions. Samples from cultures taken prior to glucose exhaustion at the diauxic shift (DS), in stationary phase (SP), & from Q & NQ cells were harvested, DNA isolated, & bar codes amplified for microarray analysis.
The resulting data was examined to determine the relative abundance of each individual strain. Three deletion pools were used; homozygous diploid, heterozygous diploid, & DAmP. The homozygous diploid deletion set, in which both alleles of the gene have been deleted, consists of the genes that have not been determined to be essential for yeast viability. The heterozygous diploid deletion set was constructed to look at those essential genes by only deleting one of the alleles in the strain so that we could examine the effect of knocking down the expression of that gene. The DAmP set (Decreased Abundance by mRNA Perturbation) is a subset of the essential genes in which the genes have not been knocked down or out, but the mRNA product from the translation process is perturbed to reduce its half-life. This inhibits the functionality of the mRNA itself & decreases the expression of the resultant protein. Since the gene is still complete in the genome it may prevent other proteins from being upregulated to mask its absence in the cell & provide a better picture of its function. The strains in the deletion sets are constructed with unique barcodes flanking a kanamycin resistance marker in place of the original open reading frame. The kanamycin marker makes the strains resistant to kanamycin. These barcodes make it possible to identify the abundance of each strain by an affymetrix style analysis.
The strain abundance data consisted of the five replicates for each of the four samples (DS, SP, NQ, Q). We only considered the 5760 strains that had measurements above the background (200) in the DS set. We primarily examined the homozygous deletion set data for determination of strains to assay. We were particularly interested in what deletions would prevent the formation of Q cells. The most significant results reveal genes important for Q cell formation, or at least the differentiation of SP cultures into NQ & Q. The most important processes that these genes & the proteins they encode are involved in seem to include mitochondria & cell organization. This corroborates what we have determined with regard to the transcriptome & proteome. We chose the 411 that were important for Q cell formation for further phenotypic analysis.
The assays provided information on the viability & levels of reactive oxygen species (ROS) in stationary phase of each strain. Viability was determined using the FungaLight test kit, consisting of two dyes. The resulting FRET interaction of the dyes results in a label that is red shifted for dead cells. Each stain is added simultaneously to a buffered solution of a relatively constant concentration of yeast cells & allowed to incubate for 30 minutes. We used the Accuri C6 flow cytometer to collect the fluorescence data. The relative abundance of ROS in each sample is determined by staining with dihydroethidium bromide. The dye incubates with the sample for 10 minutes before fluorescence data is collected. There are a number of proteins that are significantly more highly expressed in Q than NQ cells. Each strain consists of the gene for GFP fused to an individual open reading frame for each gene in yeast. We separated the strains that exhibited differential expression in SP into NQ & Q fractions.
The GFP strain CIT1:GFP was found to be a marker for Q cells. Citrate synthase is the first enzyme in cellular respiration. About 60% of the strains in the GFP library that distinguished Q from NQ cells were mitochondrial in function. The importance of mitochondrial function was further corroborated by this study on deletion strains. CIT1:GFP is expressed highly & homogenously in all Q cells, while much less is expressed in approximately 30% of NQ cells & there is none at all in the other 70% of the NQ cells. We pooled the 1000s of deletion strains together due to that particular obstacle – anytime organisms are grown in a pool there is an element of competition that is introduced. All of the results from this experiment are confounded by that variable. The relative numbers of Q & NQ cells in a stationary phase culture of CIT1:GFP is readily estimated by flow cytometry. We proposed the use of this CIT1:GFP protein marker. We selected 6 of the 411 strains for a proof of principle experiment to determine if we could create haploids with the CIT1:GFP and the deletion. This would be analogous to the homozygous diploid deletion set in that no copies of that particular gene would be present in the genome. The haploid deletion strains were mated with CIT1:GFP strain. The resulting diploids were sporulated. The tetrads of haploid spores were dissected. The colonies produced by each spore were plated onto media without histidine & with kanamycin, the resistance marker in the deletion cassette. The GFP strains have a histidine marker & are capable of growth in its absence. If the spore that produced the colonies has both of these markers, then we can conclude it has the Cit1:GFP gene as well as the deletion cassette.
Address Goals
The primary strategic goal that this research addresses is that of discovery. This research is clearly not hypothesis-driven and focuses on broadening our understanding of this particular cellular process, namely cell differentiation. Collaborative learning is also a goal clearly addressed by this research. The experimentation included researchers from multiple different backgrounds, including biology, chemistry and engineering. This is in line with the goals of NSF that seek to create a more interdisciplinary, inclusive scientific community.
