
	1999 Summer Scholars Program

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Genetic Screens of Saccharomyces cerevisiae Deletion Strains in Search of Proteins involved in TOR Signaling

By Farley C. Johnson
Mentors: Dr. Steven Zheng, Ting-Fung Chan
Department of Pathology, Washington University School of Medicine, St. Louis, MO

The laboratory which I was privileged to work in during the summer, the Zheng lab, studies the roles of TOR (Target of Rapamycin) using the budding yeast, Saccharomyces cerevisiae, as the model system.

Rapamycin is an antibiotic that was first discovered in Streptomyces hygroscopicus for antifungal activity. Higher eukaryotic cells were later proven to be affected when treated with rapamycin by their arrest at the G1 stage (first part of interphase in the cell division process) of the cell cycle. Through rapamycin causing G1 stage cell cycle arrest, a protein was discovered, TOR, when its functions were inhibited in the presence of rapamycin.

TOR is a protein that belongs to the phosphatidylinositol kinase (PIK)-like family, in which members adopt a common domain at the carboxyl terminus similar to the catalytic domain of phosphatidylinositol (PI) kineses. TOR has been shown to have a role in signal transduction pathways leading to cell cycle progression. Its other functions are unknown. TOR is found in humans and yeast as mTor (mammalian TOR or FRAP) and Tor1p and Tor2p, respectively; the mechanism of inhibition by rapamycin is similar, suggesting that TOR is well conserved throughout evolution.

A recent, ongoing project, the Saccharomyces cerevisiae Genome Deletion Project, involves the attempt to knock out all 6400 genes of budding yeast individually. So far, approximately 500 genes have been found to be essential for cellular function, which means that deletion will lead to a lethal phenotype. Half of the 6400 genes have previously been identified, leaving more than 2000 genes whose cellular function is unknown. The project will provide a powerful tool for researchers to study the remaining genes that have never been characterized.

In order to understand this approach, one must understand a genetic relationship known as synthetic lethality. When there is more than one pathway regulating a biological process, deletion of any one of the genes will show no phenotypical change because the other pathway can compensate the one that has been made defective.

My particular project presents a powerful way to observe all of the pathways that would lead to seeing all of the roles of TOR through genetic screening. By adopting the concept of the deletion project as well as understanding synthetic lethality, we hypothesized that a corresponding gene that was deleted from a particular yeast strain could be related to TOR through the involvement of its signaling pathway or biological process. My actual approach was to streak yeast strains on YPD (Yeast extract, Peptone, and Dextrose media) plates that contained 25 nM of rapamycin and YPD plates that did not contain the rapamycin. The YPD plates that did not contain the rapamycin were used as a control to see if the effect of TOR function was due to rapamycin alone. The actual experiment was observed on the YPD plates that contained rapamycin. A wild-type (WT), non-mutated strand which was not manipulated for the gene knockout deletion, was streaked on every plate that contained streaked knockout strains in order to note the difference in the cellular growth rate between the two. The expected results through observing the growth rate of colonies on plates have three possibilities. Very apparent growth on a plate implies that a knockout strain is resistant to rapamycin. No growth on the plate implies that the knockout strain is hypersensitive to rapamycin. If the results note hypersensitivity or resistance, it would suggest that the corresponding gene might somehow be related to TOR, possibly in a synthetically lethal relationship. If there is no apparent change in growth rate, the corresponding gene may not be related to TOR.

The ultimate goal is to find the knockout strain that is very apparently functioning with TOR. The next step involves the study of the genes that are somehow related to TOR in order to find possible roles of the protein within a cell, concerning signaling pathways and biological processes. This regulation of the TOR protein can lead to more insight on immunological approaches in transplantation medicine.

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