Selenium Effects on UV
Stressed Yeast’s Mutagenesis
Rate
Kris Sabatini
CCHS, Grade 10
3rd Year in PJAS
Ultraviolet Rays
- Ultraviolet (UV) rays have shorter wavelengths than
visible light. They range from 400nm to 10nm.
- Given off from the sun but most are absorbed by the
ozone layer.
- Problems in humans caused by DNA damage: sun
burn, sun poisoning, skin irritation, redness, photoaging, nausea, and possibly skin cancer.
- Shorter non-lethal exposure times can lead to
mutations.
Electro-magnetic spectrum
Selenium
-Atomic Number,34 Atomic Weight, 78.96
- Commonly taken as a supplement although
the supplement contains other components
- Considered a essential trace mineral although
it is rare to have a selenium deficiency
- A lethal dose of pure selenium 5 mg per of kg
body weight
- Has been linked to many positive health
factors such as reducing the risk of Cancer,
AIDS/HIV and Diabetes
Yeast
• Most studied cell in the world
• Easy to grow and culture
• Similar cell cycle, biochemistry and
genetics to other eukaryotic cells
• Saccharomyces cerevisiae
• The Yeast in this experiment is unable
to make the amino acid, Lysine, making it
unable to grow by itself
DNA and Mutations
- Deoxyribonucleic
acid
- Transcribed by RNA which is used by the ribosome
to ensemble proteins from Amino Acids
- Eukaryotes have DNA inside their Nuclei
-Mutations are changes made to a cell’s genome
- Can by caused by radiation, viruses, chemical
mutagens, and random errors in DNA replication
- Most mutations are harmless
a - Ketoglutarate
Lysine
AcCoA
CoA
HC
Synthase
Homocirate
Water
Homoaconitate
•Lysine’s codons are AAA and
AAG
NAD
NADH
CO2
Homoisocitrate
LYS7
LYS4
Glutamate
•There are defined minus lysine
yeast mutants used in research.
•Lys 2 mutants are missing an
enzyme function within the lysine
biosynthesis pathway.
•Result – cells require lysine
supplementation
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
Ames Test
-Developed to test the mutagenic and anti-mutagenic properties
of various chemicals by Bruce Ames in 1970s.
- Ames used a minus histidine mutant Salmonella (single point
substitution). Bacteria cannot synthesize histidine due to this
mutation.
-Exposure to suspected mutagen correlated with increased
reversion (mutation) rate.
- Visible colonies appearing on complete (-His) media evidence of
mutation through reversion
-Obviously, a lower limit on mutation rate, because only 1 DNA
site in genome assayed.
Ames Test Analogue for Yeast
- The minus lysine yeast are a result of a single substitution in
the lys-2 gene.
- A reversion at that point can result in a reversion back to wild
type yeast (lys +).
- The number of reverted colonies of yeast can be correlated
with the rate of mutation.
-This test is not test general mutation, only mutation at a single
point. The numbers are relative.
Objective/Propose
- To determine what effect
Selenium has on UV
Stressed Yeast’s
Mutagenesis Rate
Hypothesis
Null - Selenium will have no
significant effect on the
mutagenesis rate of UV
stressed Saccharomyces
cerevisiae .
Important Questions
•Was there a significant interact between the variables
of Selenium supplementation and UV Light cell
mutagenesis?
•Did the Selenium affect the mutagenesis rate of nonstressed yeast?
•Did the UV light affect the mutagenesis rate of yeast
without selenium added?
•Did the selenium affect the mutagenesis rate of 10
second exposure group?
•Did the selenium affect the mutagenesis rate on the 20
second exposure group?
Materials
• 45 (-) Lysine YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose,
1.5% agar)
•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)
• UV Hood
• Rubber Gloves
• Test tubes
• Test Tube Rack
• SDF Test Tubes
• Vitamin World Brand 200 mcg Selenium Tablets (Recommended dose of
one tablet a day) other than Selenium the Tablets contain, Dicalcium
Phosphate, Vegetable Cellulose, Brewer’s Yeast, Vegetable Stearic Acid,
Vegetable Magnesium Stearate, and Silica
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 he
cell pellet and replaced with 3 mL of SDF.
3. A selenium stock solution of 20% (total mass) was made.
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 15 mL tubes.
X = to the recommended diet intake 200 mcg/ 5 L
SDF
Selenium
Stock
Yeast
Total Volume
Tube 1 (0x)
1 mL
0 mL
1 mL
2 mL
Tube 2 (1x)
.99 mL
.01 mL
1 mL
2 mL
Tube 3 (10x)
.9 mL
.1 mL
1 mL
2 mL
7. The cells were allowed to sit for 15 min.
8. .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. 5 plates from each group were exposed to the
following UV light times, 0s, 10s and 20s.
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
Selenium Effects on UV stressed Yeast Mutagenesis Rate
40
35
# of colonies
30
25
20
0x Selenium Dose
15
1x Selenium Dose
10x Selenium Dose
10
5
0
0s
10s
Exposure Time to UV Light
20s
ANOVA Analysis
•Was there a significant interact between the variables of
Selenium supplementation and UV Light in cell mutagenesis?
•P-value - 3.38E-17, significant
•Did the Selenium affect the mutagenesis rate of non-stressed
yeast?
•P-value - 4.47E-13 , significant
•Did the UV light affect the mutagenesis rate of yeast without
selenium added?
•P-value - 8.04E-13 , significant
•Did the selenium affect the mutagenesis rate of 10 second
exposure group?
•P-value - 0.188554, insignificant
•Did the selenium affect the mutagenesis rate of the 20 second
exposure group?
•P-value - 0.003505 , significant
T crit = 4.03
Dunnett’s Test
Variable Compared
T-value
Variation, significant or
insignificant
1[x] 0s compared to
control
10[x] 0s compared to
control
T-value = 3.23
T-value = .230
Insignificant
1[x] 10s compared to
0[x] 10s
10[x]10s compared to
0x 10s
T-value = .232
T-value = 2.36
Insignificant
1[x] 10s compared to T-value = .192
0[x] 20s
T-value = 8.48
10[x] 10s compared to
0[x] 20s
Insignificant
Significant
Conclusions
•The Null Hypothesis was rejected. The selenium appeared to
have an effect on the rate of mutagenesis. ANOVA tests support
this conclusion.
•ANOVA tests appeared to support that the selenium has an
effect on the non-stressed yeast. It appeared to lower the
mutation rate in non-stressed yeast.
•The Selenium appeared to have no effect on the 10s group.
The ANOVA analysis and Dunnett’s tests supports this.
•The Selenium also appeared to have an effect on the 20s group
but only in the 10x dosage according to the Dunnett’s Test.
•The studies claim selenium has an anti -carcinogenic effect. It is
an antioxidant and may remediate or prevent molecular damage.
Limitations
•Due to the amount of cells needed to have significant
results, the amount of cells on each plate may have
differed slightly.
•The yeast cells may have needed more than 3 days to
grow completely so some colonies may not have been
visible. To compensate, the plates were rechecked after 5
days.
•When the yeast cells sat to absorb the selenium, they
may not have been allowed to sit long enough to
completely absorb it.
•The lag time between making each tube may have
caused slightly different times for plating and absorption
periods.
Further Experimentation
-Use another supplement, like the
Omega Fatty Acids
- Use higher doses of the selenium
- Use higher exposure times to UV light
- Use a different stressor