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Gene Deficiencies In Auxotrophic Yeast Strains (Saccharomyces Cerevisiae)

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Introduction

Genes are responsible for encoding the characteristics, such as phenotypes, of an organism. Genetic screening allows the function of a gene to be determined. In this experiment, a forward genetic screening approach was done to screen for mutated genes in four mutant strains of Saccha-romyces cerevisiae yeast. We mated experimental strains — wild-type (WT), strain J, strain K, strain L, and strain M — with tester strains that had a known mutation on a rich medium. After in-cubating the crosses for three days, we replica-plated the strains to a SD-ade medium plate, lacking adenine, and placed them back in the incubator. We hypothesized that the red mutant is an ade1 or ade2 mutant based on the adenine pathway since a buildup of AIR and oxidation would create red pigmentation.

We predicted that strains J, K, and M would have mutations in ADE1 or ADE2 due to their red pigmentations and that strain L would have a mutation in ADE4, ADE5, ADE8, ADE6, or ADE7 due to the cream-color and lack of red pigmentation. Results Complementation testing of yeast strains containing a wildtype yeast strand (WT) and four auxotrophic mutant strands (J, K, L, and M) was done. In the first part of the experiment, there was no growth in when strain L was combined with ade2 or when strain M was combined with ade8. There was growth on the remaining strands however. For strains J and K, growth occurred in all crosses except for ade1. For strain L, growth occurred in all crosses except for ade8. For strain M, growth occurred in all except for ade2. The yeast strains containing mutations were replicated onto an SD-ade plate. The wildtype showed complementation for all mutations. Complementation that occurred showed a thick white growth while no complementation showed no change meaning there was a clear or red color. White or cream growth indi-cates complementation has occurred. Red growth or no growth indicates no complementation has occurred. Each “+” indicates grown on adenine deficient media when the mutant strain is crossed with the tester strain. Each “-” indicates no growth. Yeast Strains WT (+) ade1 (-) ade2 (-) ade8 (-)WT (+) + + + +J (-) + – + +K (-) + – + +L (-) + + + -M (-) + + – +

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Discussion

Complementation testing allows for the determination of whether or not multiple genes are involved in a mutation creating a certain phenotype.

In our experiment, growth was seen on the adenine deficient media suggesting that those contain a mutation on that specific gene synthesizing with the media. A thick white growth was seen in crosses where complementa-tion occurred showing that it contained a mutation on the specific gene for adenine. Our results support our hypothesis and predictions based off the colors of the strains after growth. Complementation occurred in J and K with ade2 and M had complementation with ade1. Two mutations can occur in the biosynthetic pathway allowing for red and cream pig-mentation in the yeast. Red pigmentation means that the ADE1 or ADE2 enzyme has a mutation while cream pigmentation indicates that the ADE4, ADE5, ADE6, ADE7, or ADE8 enzyme has a pigmentation. If the yeast cell was blocked in one of the two steps after AIR, P-ribosylaminoimidazole, then the yeast cells turned red due to oxidation and a building up of the red pigmentation. If the yeast cell was not blocked by AIR, then the cell would be cream or white in color.

The results of the experiment show that strains J and K had an ade1 mutation be-cause no growth occurred, strain L had an ade8 mutation, and strain M had an ade2 mutation. These results support our hypothesis. Further tests using genetic screening and similar experiments to this one could be done to determine the specific location of the mutation on the gene. The results of these tests can allow bet-ter understanding about the relationship between genes and mutations in organisms. Research done with the yeast also opens up the possibility to do similar research on human genes in order to look at gene mutations that create disorders in humans.

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