Vitamin C Attenuation of Yeast Mutagenesis Peter Chekan Central Catholic High School Pittsburgh

Ultraviolet Light Electromagnetic radiation Produced by the sun Wavelength shorter than that of visible light Greater energy, thus higher risk Can cause sunburn and skin cancer Damages DNA

Oxidative Stress UV light can result in oxidation stress on cells Caused by an imbalance in the production of reactive oxygen Increases oxidant production in cells Results in cellular degeneration Could cause various cancers, such as skin cancer Antioxidants are thought to counter oxidative stress

Antioxidants Molecules capable of slowing or preventing the oxidation of other molecules May be able to prevent cancer and coronary heart disease Body produces antioxidants Can obtain through Diet Vitamin C

Antioxidant Enzyme cofactor Also known as Ascorbic acid In Oranges, Strawberries, and Grapefruit Recommended daily intake: 60 mg The disease scurvy occurs from lack of Vitamin C

Saccharomyces cerevisiae(Yeast) Saccharomyces cerevisiae (Yeast) Eukaryotic microorganism Unicellular, 3–4 µm diameter Used in baking and production of alcoholic beverages Cell cycle is similar to human cells Comparable DNA replication, recombination, cell division and metabolism The most studied cellular model in research (-) lysine mutant (-) lysine mutant can be used to explore mutagenesis

Lysine Codons: AAA, AAG Essential Amino Acid  Not able to be produced by human Responsible for Calcium absorption, building muscle protein and body's production of hormones Essential for the growth of Saccharomyces cerevisiae

Lysine a - Ketoglutarate AcCoA CoA HC Synthase Homocirate LYS7 Water Homoaconitate Homoisocitrate a - Ketoadipate a- Aminoadipate a- Aminodipate Semialdehye SaccharopinaLysine LYS4 LYS12 aAA- Aminotranfease LYS2 LYS9 LYS1 NAD NADH CO 2 Glutamate a-Ketoglutrate ATP PP NADPH NADP Glutamate NADPH NADP Water NADP; NADP a- Ketoglutarate

DNA Mutations Changes in DNA sequence of a cell's genome radiation Caused by radiation, viruses, mutagenic chemicals, and errors during DNA replication Point Mutation Types: Insertion, Deletion, Frameshift, Point Mutation  Replacement of a single base nucleotide with another nucleotide of the genetic material  Sickle cell anemia

ReverseMutation Reverse Mutation Also called Reversion and Back Mutation A mutated gene mutates back to the wild-type phenotype  called revertants

Ames Test Created by Bruce Ames Biological assay used to assess the mutagenic potential of a chemical Reversion rate of –His to +His used to assess mutagenesis. Positive test indicates chemical carcinogen  Positive- greater number of colonies than control Saccharomyces cerevisiae Saccharomyces cerevisiae will be used as the model instead of Salmonella typhimurium

Modified Ames Test (Yeast) A lys (-) strain of yeast employed: cannot synthesize lysine due to single point mutation Revertable Complete (-lys) media plates used to assay for reversion

Purpose Determine if the antioxidant Vitamin C can reduce the mutagenesis rate of UV-stressed cells

Hypothesis Null Hypothesis The vitamin C concentrations will not significantly affect the UV-stressed yeast mutagenesis rate Alternative The vitamin C concentrations will significantly affect the yeast mutagenesis rate

Materials Sterile conical tubes  15 ml  50 ml Test tube rack Micropipette Pipette tips Yeast (Saccharomyces cerevisiae)  minus lys Complete (minus lys) agar Plates Vitamin C  Proper safety equipment  Sterile Water  Spreader bar  Vortex  Sterile dilution fluid  UV Safety Glasses  Incubator  Ethanol  Matches  UV Lamp/hood

Procedure 1. A strain of yeast (-) Lys phenotype was grown for 2 days in YEPD media 2. 1 day prior to experimentation the media was removed and the cell pellet washed with SDF 3. The pellet in SDF was resuspended 4. The following ingredients were pipetted into sterile 15 mL tubes

Procedure 5. The cells were allowed to incubate for 15 min 6. The yeast was resuspended mL aliquots were spread onto 45 complete (-) Lys agar plates (necessary to define cells that have reverted through mutation to wild type (+) lys ) 8. 5 plates from each group were exposed to the following exposures of UV light: 0s, 10s and 20s 9. The plates were incubated at 32 ºC for 3 days 10. Revertant colonies were counted and recorded.

Data

P=7.86E-09 P<0.05 P=3.02E-07 P<0.05 P=1.07E-06 P<0.05 P>0.05 P=5.24E-21 P<0.05

Dunnett Test Result

Interpretations Statistical Analyses suggests that UV light significantly affected yeast with or without Vit C Statistical Analyses supports no significant variation caused by Vitamin C Statistical Analyses suggests no interaction between variables

Extensions Use different concentrations of Vitamin C Use different types of antioxidants (Lycopene, Vitamin A, Vitamin E) Expose to varying amounts of UV light Increase sample size Synchronize cell plating times more effectively

Conclusion Accepted The statistical analyses allows the null hypothesis to be Accepted, indicating that vitamin C did not significantly effect the mutantion rate for yeast The UV light had a significant effect on Saccharomyces cerevisiae  Supports that UV light causes mutation

References es.jpg es.jpg nC/ nC/ ml ml Dr. Wilson, biostatistician, University of Pittsburgh