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.

Data

P=7.86E-09P<0.05

P=3.02E-07P<0.05

P=1.07E-06P<0.05

P>0.05

P>0.05 P>0.05P=5.24E-21P<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

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