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lab3fulllab - UV mutagenesis and mutagenic rates for de...

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UV mutagenesis and mutagenic rates for de novo Lys2 and Lys5 yeast auxotrophs via alpha-aminoadipic acid selection Abstract UV mutagenesis and mutagenic rates for de novo Lys2 and Lys5 yeast auxotrophs via alpha-aminoadipic acid selection
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Biochemical analysis has previously led to the belief that a-aminoadipic acid is nitrogen source because it is part of a pathway for formation of glutamic acid, however, when yeast is simply plated onto minimal media with aaa, little to no growth occurs. Another pathway utilizes aaa for lysine production, and eventually poisons the cell with byproducts. Lysine auxotrophs can utilize aaa as a nitrogen source, as long as they are supplemented with lysine because the lysine pathway no longer poisons the cell. Here I studied the genetic basis for the different pathway mutations yielding lysine auxotrophs, and analyzed the effect of UV exposure to mutagenic rates and mutant properties. I isolated 10 UV exposed and 10 unexposed lysine auxotrophs using an aaa media as a genetic selection pressure, then preformed complementation testing with known lys2- and lys5- yeast strains. I found spontaneous mutation yielded 6 lys2-, 0 lys5-, 2 double mutants, and 2 unknown genotype colonies. I also found that UV exposure yielded 2 lys2-, 5 lys5-, 1 double mutant and 2 unknown genotype colonies. I determined that aaa selects mainly for lys2 mutants, UV mutagenesis increases the mutagenic frequency, and increases the mutational rate of lys5 by a greater extent than that of lys2 and that there were lysine auxotrophs with incomplete loss of function mutations, or mutation in genes besides lys2 or lys5. Future experiments will specifically determine the causes for these events by base pair sequencing. Introduction Wild type yeast cells grow on medium containing various chemicals and nutrient sources because yeast synthesize complex cellular components from simpler substances. In particular yeast can synthesize most amino acids from basic starting materials; here the important synthesizable amino acid is lysine. To generate these cellular components yeast requires (among
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other nutrients) a source of nitrogen. Three different substances can be utilized as a nitrogen source, ammonium sulfate, glutamic acid, and in certain circumstances alpha-aminoadipic acid (here aaa) (Larson et al, 1962 ) . Biochemical analysis has previously led to the belief that aaa is nitrogen source because it is part of a pathway for formation of glutamic acid, since it can form a known nitrogen source (glutamic acid) a nitrogen supply is available to the yeast cell (Kosinski- Collins, 2006). However, when yeast is simply plated onto minimal media with the only nitrogen source being aaa, little to no growth occurs. The absence of large growth can be attributed to a deviation from the aaa to glutamic acid pathway resulting in negative side effects. In wild type yeast there is an overabundance of a-aminoadipic acid reductase (EC 1.2.1.31, or L- aminoadipate-semialdehyde dehydrogenase, here called aad), an enzyme that converts the aaa into an intermediate product for the lysine production pathway. When this pathway is activated,
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