Vitamin C Attenuation of Yeast Vitamin C Attenuation of Yeast MutagenesisMutagenesis
Peter ChekanPeter Chekan
Central Catholic High School Central Catholic High School
PittsburghPittsburgh
Ultraviolet LightUltraviolet 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 StressOxidative 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 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
Vitamin CVitamin 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 cerevisiaeSaccharomyces cerevisiae (Yeast)(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
LysineLysine
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
AcCoACoA
HCSynthase
Homocirate
LYS7Water
Homoaconitate
Homoisocitrate
a - Ketoadipate
a- Aminoadipate
a- AminodipateSemialdehye
Saccharopina Lysine
LYS4
LYS12
aAA- Aminotranfease
LYS2
LYS9
LYS1
NADNADHCO2
Glutamatea-Ketoglutrate
ATPPPNADPHNADP
GlutamateNADPHNADPWater
NADP; NADPa- Ketoglutarate
DNA MutationsDNA Mutations
Changes in DNA sequence of a cell's genome Caused by radiationradiation, viruses, mutagenic
chemicals, and errors during DNA replication Types: Insertion, Deletion, Frameshift, Point Point
MutationMutation Replacement of a single base nucleotide with
another nucleotide of the genetic material Sickle cell anemia
ReverseReverse MutationMutation
Also called Reversion and Back Mutation A mutated gene mutates back to the wild-type
phenotype called revertants
AmesAmes TestTest 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 cerevisiaeSaccharomyces 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
PurposePurpose
Determine if the antioxidant Vitamin C can reduce the mutagenesis rate of UV-stressed cells
HypothesisHypothesis
Null HypothesisNull Hypothesis The vitamin C concentrations will not
significantly affect the UV-stressed yeast mutagenesis rate
AlternativeAlternative The vitamin C concentrations will
significantly affect the yeast mutagenesis rate
Materials 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
7. 0.1 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.
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
ExtensionsExtensions
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
ConclusionConclusion
The statistical analyses allows the null hypothesis to be AcceptedAccepted, 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
http://www.bruceames.org/ http://davidmlane.com/hyperstat/B112114.html http://en.wikipedia.org/wiki/Vitamin_C http://en.wikipedia.org/wiki/
File:Ambersweet_oranges.jpg http://lpi.oregonstate.edu/infocenter/vitamins/
vitaminC/ http://davidmlane.com/hyperstat/B112114.html http://www.nlm.nih.gov/medlineplus/
antioxidants.html Dr. Wilson, biostatistician, University of Pittsburgh
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