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Utilizing yeast genetics to identify novel genes involved in translation fidelity

Samanfar B, Golshani A: Utilizing yeast genetics to identify novel genes involved in translation fidelity. 63rd Annual Conference of the Canadian Society of Microbiologists (CSM), 2013, Ottawa, Canada.

Abstract

Predicting gene functions is a major goal of systems molecular biology in the post genomic era. In this context the yeast, Saccharomyces cerevisiae, has emerged as one of the few model organisms of choice for large-scale functional genomics investigations. Protein synthesis or translation is an essential process within a cell with significant implication in the etiology of human diseases like cancer. Having a better understanding of translation process and characterizing novel genes involved in this pathway is the focus of a number of current investigations. In order to systematically identify novel genes that affect the fidelity of translation 3 plasmids (pUKC817, pUKC818 and pUKC819) with different premature stop codons within LacZ expression cassette along with a fourth plasmid (pUKC815), used as a control, were transferred into yeast non-essential gene-deletion array through a modified Synthetic Genetic Array (SGA) method for a total of 20,000 strain transformations. Large scale β-galactosidase lift assay followed by quantitative low-throughput β-galactosidase assay was performed. In this way 84 potential candidate genes that affect translation fidelity were selected. 8 candidate genes were followed up and analyzed for their involvement in translation efficiency using inducible plasmid based β-galactosidase assay followed by qRT-PCR and drug sensitivity spot test analysis. Our SGA and Synthetic Dosage Lethality analysis for 4 candidate genes indicated the involvement of BSL1, BSL2 and BSL3 in ribosome biogenesis and BSL4 in amino acid biosynthesis. A surprising observation was that a number of cell wall associated genes also reduced translation fidelity confirming a previously reported connection between cell wall and translation.

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