Advances in DNA sequencing have provided the complete genome sequence for many organisms. However, understanding the functions of each gene product encoded by the genome remains a major challenge. This dissertation describes an improved strategy for identifying functional relationships between genes and the use of unbiased genetic and biochemical approaches to uncover the function of the ORM family of genes.
Large-scale measurement of genetic interactions has proven to be a powerful approach for functional genomics in the model organism Saccharomyces cerevisiae. The key to such analyses is comparing the effects of mutations to individual genes or pairs of genes on a phenotype of interest, such as growth rate. In the first part of my thesis, I developed a high-throughput, flow cytometry-based strategy for measuring growth rates in S. cerevisiae with improved accuracy. This assay made it possible to precisely quantify the contribution of each gene to growth and to measure genetic interactions with increased sensitivity. With high-precision data, I was able to resolve an enigma regarding the apparent prevalence of gene dispensability in yeast, to reveal new relationships between chromatin modifying factors, and to create a functional map of the proteasome.
In the second part of my dissertation, I used genetic interactions as a starting point to identify members of the conserved ORM gene family as negative regulators of sphingolipid synthesis. I found that Orm proteins form a complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production, and that a phosphorylation-based regulatory pathway relieves the inhibitory activity of Orm proteins when sphingolipid production is disrupted. This feedback mechanism matches sphingolipid supply to cellular demand, with changes in ORM gene expression or mutations to their phosphorylation sites causing dysregulation of sphingolipid metabolism. Thus, given the essential roles of sphingolipids as structural components of membranes and as signaling molecules, Orm proteins serve a key role in cellular homeostasis. Finally, the functional conservation within this gene family and the recent identification of the human homolog ORMDL3 as a potential risk factor for childhood asthma raise the possibility that sphingolipid misregulation contributes to asthma development.
