HIGH-THROUGHPUT GENOMIC/PROTEOMIC STUDIES, FINDING STRUCTURE AND MEANING BY SIMILARITY (2010)

The post-genomic challenge was to develop high-throughput technologies for measuring genome scale mRNA expression levels. Analyses of these data rely on computers in an unprecedented way to make the results accessible to researchers. My research in this area enabled the first compendium of microarray experiments for a multi-cellular eukaryote, Caenorhabditis elegans. Prior to this research approximately 6% of the C. elegans genome had been studied, and little was know about global expression patterns in this organism. Here I cluster data from 553 different microarray experiments and show that the results are stable, statistically significant and highly enriched for specific biological functions. These enrichments allow identification of gene function for the majority of C. elegans genes. Tissue specific expression patterns are discovered suggesting the role of particular proteins in digestion, tumor suppression, protection from bacteria and from heavy metals. I report evidence that genome instability in males involves transposons, and find co-expression patterns between sperm proteins, protein kinases and phosphatases suggesting that sperm, that are transcriptionally inactive cells, commonly use phosphorylation to regulate protein activities. My subsequent research addresses protein concentrations and interactions, beginning with a simultaneous comparison of multiple data sets to analyze Saccharomyces cerevisiae gene-expression (cell cycle and exit from stationary phase/G0) and protein-interaction studies. Here, I find that G1-regulated genes are not co-regulated during exit from stationary phase, indicating that the cells are not synchronized. The tight clustering of other genes during exit from stationary-phase does indicate that the physiological responses during G0 exit are separable from cell-cycle events. Subsequently, I report in vivo proteomic research investigating population phenotypes in stationary phase cultures using the yeast Green Fluorescent Protein-fusion library (4156 strains) together with flow cytometry. Stationary phase cultures consist of dense quiescent (Q) and less dense non-quiescent (NQ) fractions. The Q-cell fraction is generally composed of daughter cells with high concentrations of proteins involved in the citric acid cycle and the electron transport chain, for example Cit1p. The NQ fraction has subpopulations of cells that can be separated by the low and high concentrations of these mitochondrial proteins, i.e., NQ cells often have double intensity peaks: a bright fraction and a much dimmer fraction, which is the case for Cit1p. The Q fraction uses oxygen 6 times as rapidly as the NQ fraction, and 1.6 times as rapidly as exponentially growing cells. NQ cells are less reproductively capable than Q cells, and show evidence of reactive oxygen species stress. These phenotypes develop as early as 20-24 hours after the diauxic shift, which is as early as we can make a differentiating measurement using fluorescence intensities. Finally, I propose a new way to analyze multidimensional flow cytometry data, which may lead to better understanding of Q/NQ cell differentiation.