BIO LAB URA YEAST

BIO LAB URA YEAST - Identification and Analysis of...

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Identification and Analysis of Temperature Sensitive Conditional Mutations in the Saccharomyces cerevisiae Ura3 Protein Using FOA Selective Genetic Screen By John Domanick and Ayush Pathak
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ABSTRACT Analysis of Temperature Sensitive Mutations of Ura3p in S. cerevisiae J. Domanick and A. Pathak, Department of Biology, Brandeis University
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It is very difficult to study the actions of essential genes in organisms, especially haploid organisms such as Saccharomyces cerevisiae , due to the often result of death from mutations of essential genes. Conditional mutations, such as temperature sensitive mutations, are a very useful tool in studying these genes, and they can be turned on and off by a change of environment. We investigated temperature sensitivity mutations in URA3 of S. cerevisiae , and the structure of said mutations compared to wild type S. cerevisiae . We selected for mutants on FOA selective media, then moved mutants to uracil dropout plates, set in three different environmental temperatures. Any mutants found to grow on both FOA and low temperature uracil dropout could possibly be a ts mutant we were looking for. We found a S. cerevisiae specimen containing an A107P mutation in Ura3p, pluss two silent mutations also within the URA3 gene. This specimen grew on FOA, but failed to grow on uracil dropout at any temperature. The mutation A107P in URA3 does not lead to a temperature sensitive Ura3p protein, but leads to a complete loss of function protein. Further studies will assess the reason A107P causes a complete loss of function and not a temperature sensitive mutation. INTRODUCTION Genes are sequences of DNA that code for the production for specific proteins. Every three nucleotide bases of DNA codes for the incorporation of a specific amino acids into the protein primary structure. This means that mutations, or changes, in the DNA sequence may lead to the incorporation of new amino acids into the protein, which changes its structure, and thereby changes its functionality. These mutant proteins can be totally ruined, and lose function, be mostly unaffected, and retain functionality, or be altered just enough to function under certain conditions, while becoming nonfunctional under other conditions. These types of mutations lead to what is called a conditional mutant organism, one which appears wild type in one setting, but may appear mutant in another. (Kosinski-Collins 2010) We screened a culture of Saccharomyces cerevisiae ( S. cerevisiae ) to identify and characterize a type of conditional mutation called a temperature sensitive (TS) mutation. A TS mutation is one that causes a protein to be functional under normal temperatures, (30 o C for our S. cerevisiae ), but dysfunctional at a higher temperature (37 o C).(Pizzagalli et al 1988) More specifically we were searching for a TS mutation of orotidine-5’phosphate decarboxylase (ura3p), an enzyme which functions in the production of uracil. Since Ura3p functions in the production of uracil, and uracil is needed for RNA production, the prevalence of mutations in
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BIO LAB URA YEAST - Identification and Analysis of...

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