Transcript vit C effects on yeast mutagenesis Chekan PJAS 2010

Vitamin C Attenuation of
Yeast Mutagenesis
Peter Chekan
Central Catholic High School
Pittsburgh
Ultraviolet Light
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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
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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
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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 C
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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)
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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 can be used to explore
mutagenesis
Lysine
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Codons: AAA, AAG
Essential Amino Acid
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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
a - Ketoglutarate
Lysine
AcCoA
CoA
HC
Synthase
Homocirate
Water
Homoaconitate
NAD
NADH
CO2
Homoisocitrate
LYS7
LYS4
Glutamate
LYS12
a-Ketoglutrate
a - Ketoadipate
ATP
PP
NADPH
NADP
aAA- Aminotranfease
a- Aminoadipate
Glutamate
NADPH
NADP
Water
LYS2
a- Aminodipate
Semialdehye
NADP; NADP
a- Ketoglutarate
Lysine
LYS9
Saccharopina
LYS1
DNA Mutations
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Changes in DNA sequence of a cell's genome
Caused by radiation, viruses, mutagenic
chemicals, and errors during DNA replication
Types: Insertion, Deletion, Frameshift, Point
Mutation
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Replacement of a single base nucleotide with
another nucleotide of the genetic material
Sickle cell anemia
Reverse Mutation
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Also called Reversion and Back Mutation
A mutated gene mutates back to the wild-type
phenotype
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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
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Positive- greater number of colonies than control
Saccharomyces cerevisiae will be used as the
model instead of Salmonella typhimurium
Modified Ames Test (Yeast)
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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
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Determine if the antioxidant Vitamin C
can reduce the mutagenesis rate of
UV-stressed cells
Hypothesis
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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
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Sterile conical tubes
 Proper safety equipment
 15 ml
 Sterile Water
 50 ml
 Spreader bar
Test tube rack
 Vortex
Micropipette
 Sterile dilution fluid
Pipette tips
 UV Safety Glasses
Yeast (Saccharomyces cerevisiae)
 Incubator
 minus lys
 Ethanol
Complete (minus lys)
 Matches
agar Plates
 UV Lamp/hood
Vitamin C
Procedure
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A strain of yeast (-) Lys phenotype was grown
for 2 days in YEPD media
1 day prior to experimentation the media was
removed and the cell pellet washed with SDF
The pellet in SDF was resuspended
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=5.24E-21
P<0.05
P=3.02E-07
P<0.05
P=7.86E-09
P<0.05
P=1.07E-06
P<0.05
P>0.05
P>0.05
P>0.05
Dunnett Test Result
Interpretations
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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
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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
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The statistical analyses allows the null
hypothesis to be Accepted, indicating that
vitamin C did not significantly effect the
mutantion rate for yeast
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The UV light had a significant effect on
Saccharomyces cerevisiae
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Supports that UV light causes mutation
References
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http://www.bruceames.org/
http://davidmlane.com/hyperstat/B112114.html
http://en.wikipedia.org/wiki/Vitamin_C
http://en.wikipedia.org/wiki/File:Ambersweet_orang
es.jpg
http://lpi.oregonstate.edu/infocenter/vitamins/vitami
nC/
http://davidmlane.com/hyperstat/B112114.html
http://www.nlm.nih.gov/medlineplus/antioxidants.ht
ml
Dr. Wilson, biostatistician, University of Pittsburgh