Vitamin E's Effects on Yeast's Mutagenesis Rate
Kristopher Sabatini
12th Grade at CCHS
5th Year at PJAS
Previous Studies
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Mixed and contradictory studies
Positive anticancer effects on bladder and
breast cancer
No effect on kidney, lung nor cardiac cancer
Linked to increase in prostate cancer in high
doses
Overall – beneficial to general health
Vitamin E
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Common Anti-oxidant
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Found in nut oils and green vegetables
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Lipid Soluble and hydrophobic
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Variety of Isomers
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Alpha and gamma tocopherols– most useful to
humans
Ultraviolet Rays
Light waves that have shorter wavelengths, thus greater
energy, than visible light
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They range from 400nm to 10nm
Given off from the sun but most are absorbed by the
ozone layer
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Mutagen – Direct DNA Damage
Yeast
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Commonly used model
Tolerant and safe to culture
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Has similar reproduction, metabolism, and
chemistry as other more advanced eukaryotic
cells
• Saccharomyces cerevisiae
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Special Strain, unable to produce Lysine
a - Ketoglutarate
Lysine
AcCoA
CoA
Homocirate
Water
Homoaconitate
Lysine’s codons are AAA and AAG
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There are defined minus lysine yeast
mutants used in research.
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NAD
NADH
CO2
Homoisocitrate
HC
Synthase
LYS7
LYS4
Glutamate
LYS12
a-Ketoglutrate
a - Ketoadipate
Lys 2 mutants are missing an enzyme
function within the lysine biosynthesis
pathway.
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Result – cells require lysine
supplementation
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ATP
PP
NADPH
NADP
aAA- Aminotranfease
a- Aminoadipate
Glutamate
NADPH
NADP
Water
LYS2
a- Aminodipate
Semialdehye
NADP; NADP
a- Ketoglutarate
Lysine
LYS9
Saccharopina
LYS1
Ames Test
Developed to test the mutagenic and anti-mutagenic properties of
various chemicals by Bruce Ames in 1970s.
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Ames used a minus histidine mutant Salmonella (single point
substitution). Bacteria cannot synthesize histidine due to this
mutation.
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Exposure to suspected mutagen correlated with increased reversion
(mutation) rate.
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Visible colonies appearing on complete (-His) media evidence of
mutation through reversion
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Obviously, a lower limit on mutation rate, because only 1 DNA site in
genome assayed.
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Modified Ames Test
(-) Lys Yeast – Eukaryote
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The number of reverted colonies of yeast can be
correlated with the rate of mutation.
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A reversion at that point can result in a reversion
back to wild type yeast (lys +).
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Mutagen substitution – UV light instead of heat
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Objective
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To determine what effects Vitamin
E has on Yeast's Mutagenesis
Rate
Hypothesis
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Vitamin E will not have an effect on Yeast's
Mutagenesis Rate
Materials
45 (-) Lysine YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar)
•UV Light Oven (LD-50 on Yeast is 30 seconds)
•Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, .1mM CaCl2,
100mM NaCl)
•Klett spectrophotometer
•Sterile pipette tips and Micropipettes
•Vortex
• Sidearm flask
•Spreader bar
•Ethanol
• Micro burner
• (-) Lysine Saccharomyces cerevisiae (John Wolford lab, CMU)
•Rubber Gloves
• Test tubes
•Microtubes
• Test Tube Rack
• SDF Test Tubes
• Vitamin E Oil Solution
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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 from the cell
pellet and replaced with 3 mL of SDF.
3. The Vitamin was sterile filtered
4. The stock solution was sterilized with a 0.22 syringe micron-filter.
5. The pellet in SDF was resuspended.
6. The following ingredients were pipetted into sterile mircotubes.
(Percents are by volume compared to stock solution)
Water
Variable
Yeast
Volume
Tubes 1 and
2
0. 8 mL
0 mL
0.2 mL
1 mL
Tubes 3 and
4
0.79 mL
0.01 mL
0.2 mL
1 mL
Tubes 5 and
6
0. 7 mL
0.1 mL
0.2 mL
1 mL
Two Tubes per concentration were used as 1.5 mL
were needed
7. The cells were allowed to sit for 15 min.
8. 0.1 mL aliquots were spread onto 45 complete (-) Lys (15
each) agar plates (necessary to show cells that have reverted
through mutation to wild type + lys ).
9. The plates were exposed to UV light for either 0, 15, 30
seconds
10. The plates were allowed to incubate for 3 days at 32o C.
11. The colonies were counted and recorded. Each colony
assumed to have arisen from 1 cell.
Results
Vitamin E's Effects on Yeast's Mutagenesis Rate
45
40
35
30
0.00%
1.00%
10.00%
25
Colonies
20
15
10
5
0
0s
15s
Exposure Time
30s
ANOVA Statistical Analysis
Alpha Value = 0.05
Test
P-Value
Interpretation
Two Factor ANOVA
0.0000239948038879 Significant
2
Single Factor ANOVA 1.49668808058975E Insignificant
on isolated on UV
-08
Single Factor ANOVA 0.806861423820598 Significant
on isolated Vitamin E
Dunnett's Test
Test
T-Value
Interpretati
on
0s 0% vs 0s 1%
1.46
Insignificant
0s 0% vs 0s 10%
1.65
Insignificant
15s 0% vs 15s 1%
8.36
Significant
15s 0% vs 15s 10%
11.98
Significant
30s 0% vs 30s 1%
6.78
Significant
30s 0% vs 30s 10%
6.56
Significant
T- crit = 3.29
Conclusion
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The Null Hypothesis can be rejected in both the 1% and
10% groups
The insignificant variance in the no exposure group fits
expectations (no mutagen thus little mutation)
In each of the two other exposure groups, the Vitamin
yielded significant results when compared back to the
control of that group
The lower colony counts in the 30s groups is may be due
to UV light outright killing the colonies
This results can be contributed to Vitamin E's anti-oxidant
abilities combating the free radicals formed by UV stress
Limitations and Extensions
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Limitations
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Unsynced plating which leads to slightly different exposure times
to Vitamin E
Inability to control the exact amount of cells on each plate (minor
difference overshadow by massive amount of cells)
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Slight positioning differences in UV Oven
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Inability to account for cell deaths due UV Light
Extensions
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Different model
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Reduce lag time with lab hands
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Almar Blue Assay to account for cell deaths
Sources
http://www.uic.edu/classes/phar/phar332/Clinical_Cases/vitamin%20cases/vitamin%2
0E/Vitamin%20E%20Chemistry.htm
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AmesTest.html
http://theoncologist.alphamedpress.org/content/6/3/298.full
http://student.ccbcmd.edu/~gkaiser/biotutorials/protsyn/mutate.html
http://jama.ama-assn.org/content/306/14/1549
http://ods.od.nih.gov/factsheets/vitamine/
http://www.huffingtonpost.com/craig-cooper/vitamine-e-prostate-cancer_b_1016922.html