Characterizing gene functions with an overexpression ORF collection in Saccharomyces cerevisiae
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My research project aims to discover new eukaryotic gene functions using yeast as a model organism. Yeast (Saccharomyces cerevisiae), are a prototypical eukaryotic model because of its high degree of genetic similarity to humans, fast generation time, and relatively low-cost maintenance. Identifying gene function in eukaryotes, such as humans, is an important, broad step in mapping gene and protein networks, predicting phenotypes, and understanding disease causation. This project uses a gain-of-function approach to characterizing new gene functions. We use a pool of transformed yeast with each yeast cell “over-expressing” a single gene carried by a plasmid, such that the quantity of the protein product encoded by the gene increases. The pool contains yeast transformed with plasmids representative of approximately 93% of all yeast genes. A plasmid is an exogenous piece of DNA that can be inserted into cells and engineered to carry specific genes of interest. Each plasmid in this pool has a galactose promoter that regulates the expression of a target gene, and only in the presence of galactose is that gene constitutively expressed. The yeast is treated with galactose to induce overexpression of a specific gene. This project aims to induce overexpression in a pool of yeast and monitor the change in abundance of each plasmid on a genome-wide scale using DNA microarrays. A DNA microarray measures the activity of thousands of yeast genes using the knowledge of complementary binding between nucleotides. By analyzing the patterns in which sets of plasmids are enriched and which drop out during overexpression, their functions may be inferred and characterized. Another aim of this project is to troubleshoot the 4 overexpression screen: from growing the yeast, to isolating the DNA from the yeast cells, and to amplifying the plasmid DNA. After several troubleshooting experiments, we found the PCR yield for yeast grown in galactose to be lower than that of yeast grown in glucose and yeast grown in raffinose. Additionally, PCR yield decreased the longer yeast grew in galactose, such that 12 hours of galactose induction yielded the lowest PCR. This could be due to the overexpression plasmids being lost from the yeast cells over time, the plasmids undergoing recombination with the genome, or plasmid DNA being lost or degraded during the DNA prep or PCR procedures. Our results also show changes in relative abundances of plasmid DNA as yeast change media from raffinose to galactose. Additionally, we found functional enrichment of distinct gene sets in yeast pools grown in raffinose and yeast pools grown in galactose. This illustrates the value this overexpression screen has in sorting genes into their functional networks, which in turn provides information in characterizing genes of unknown function.