 | Janice Sotomayor Pontifical Catholic University of Puerto Rico General ScienceMentor(s)Terri Kinzy, Ph.D. Arjun Sasikumar Department of Molecular Genetics, Microbiology and Immunology UMDNJ-Robert Wood Johnson Medical School |
| ||
| Identification of genes that interact with the eukaryotic elongation factor 3 in Saccharomyces cerevisiae | ||
| Over the past years there has been a high incidence of people who die from fungal infections. Most of the limited number of drugs available to treat these types of infection have either harmful side effects or are susceptible to resistance. Eukaryotic translation elongation factor 3 (eEF3) is a protein of interest for scientists looking for a novel specific target to fight fungal infections. eEF3 is an essential fungal protein that is involved in removing deacylated tRNA from the ribosomal E site during translation elongation. This factor is of specific interest in yeast since it is a protein with no known homologue in mammalian systems, making it a candidate for fungal specific drug target. In order to better understand the function of eEF3 we are performing a genetic screen using a strain of the yeast, Saccharomyces cerevisiae, expressing eEF3 P915L, a mutant form of eEF3 in which amino acid proline is changed to leucine. This strain, which is temperature-sensitive and unable to grow at 35°C, will be used to perform a high copy suppressor screen to identify proteins that interact with eEF3. We will transform TKY1495 (eEF3 P915L) with a high-copy yeast DNA library, containing all the yeast genes. Transformants will be selected based on their ability to grow on media plates lacking uracil since the plasmid library has URA3 as a marker. Once they grow at 30°C, 2 copies of each plate will be produced by replica plating, one of which will be incubated at 30°C and the other at 35°C. The colonies that grow at 35°C are the ones that potentially have the gene which interacts with the protein synthesis apparatus of the cell. We will verify the suppression by isolating the plasmid and retransforming TKY1495 to get colonies that can still grow at 35°C. The high-copy suppressor plasmids so obtained will be sequenced to identify the genes involved in suppression. Based on the genes identified, further experiments will be designed to characterize the interaction between them and eEF3. |