Selenium Effects on UV
Stressed Yeast Mutagenesis
Noah Stickel
Grade 10, CCHS
2nd Year in PJAS
Selenium
•
Atomic number 34, Atomic weight 78.96
•
Taken commonly as a dietary supplement
•
Considered an essential trace element, but deficiencies
are rare
•
5 mg of pure selenium per kg of weight is a lethal dose
•
There are conflicting studies regarding its antimutagenic properties
Question
•
Do Selenium supplements
significantly affect the
mutagenesis rate of yeast?
Saccharomyces cerevisiae
•
Commonly known as yeast
•
Easy and safe to grow and
culture, most commonly studied
eukaryotic cell
•
The yeast in this experiment is
unable to produce Lysine
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 the cell's genome, and are
mostly harmless
•
Can be caused by radiation, viruses, chemical mutagens, and
random errors in DNA replication
Lysine
•
Codons are AAA and AAG
•
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
Ames Test
•
Developed to test the mutagenic and anti-mutagenic
properties of various chemicals by Bruce Ames in the
1970's
•
Ames used a minus histidine mutant of salmonella,
which could not produce histidine due to mutation
•
Exposure to suspected mutagen correlated which
increased reversion (mutation) rate
•
Visible colonies appearing on complete -His media
evidence of mutation through reversion.
Modified Ames Test
•
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 does not test general mutation, only
mutation at a single point. The numbers are relative
Ultraviolet Rays
•
Ultraviolet (UV) Rays have shorter wavelengths than visible light.
They range from 400nm to 10nm
•
Given off by sun, but most are absorbed by the Ozone Layer
•
Problems in humans caused by over exposure: sunburn, sun
poisoning, skin irritation, nausea, photo-aging, and possibly skin
cancer
•
Shorter exposures can cause mutation instead of DNA damage
Purpose
•
To determine the effects of a supposed antimutagen, Selenium, on the mutagenesis rate of ()Lys yeast, using a stressor, UV light.
•
To determine the effects of a known mutagen, UV
light, on the mutagenesis and survivorship of wild
type yeast and a (-) Lys strain
Hypotheses
•
Null: Selenium will not have a
significant effect on the
mutagenesis rate of UV stressed
yeast.
•
Alternative: UV light will
significantly effect the
mutagenesis rate of yeast
Materials
• (-) 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 (CMU)
• UV Hood
• Rubber Gloves
• Test tubes
• Test Tube Rack
• SDF Test Tubes
• Giant Eagle 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,
approximately 10^8 cells per ml
2. 4 days prior to experimentation the media was removed from the cell pellet
and replaced with 3 mL of SDF. Prior to removal, a series of washes were
conducted with SDF to remove any residual lysine nutrients
3. The pellet in SDF was resuspended, and stored in (-) Lys media for 2 days
4. A selenium stock solution of 1% (total mass) was made, along with a 0.1%
stock for the 0.0001% concentration
5. The pellet in SDF was resuspended, and the following ingredients were
pipetted into sterile 1.5 mL tubes
Concentration
Microbe
Sterile Water
Stock Solution
Final Volume
0%
0.2 ml
0.8 ml
-
1 ml
0.0001%
0.2 ml
0.799
0.001
1 ml
0.1%
0.2 ml
0.7
0.1
1 ml
Procedure Continued
7. The cells were allowed to sit for 30 min.
8. 4 0.1 ml and 3 0.2 ml aliquots per experimental group
were spread onto complete (-) Lys agar plates
9. 7 plates from each concentration were exposed to the
following UV light times: 0s, 10s and 20s.
10. A UV survivorship curve of both lysine + and - was also
plated and exposed, with no additional variable
11. The plates were allowed to incubate for 5 days at 32o C.
12. The colonies were counted and recorded. Each colony
assumed to have arisen from 1 cell.
Procedure for UV Exposure
(-) Lys Yeast
1.
1.
0.1 mL of suspended Saccharomyces
cerevisae cells were plated on each of
12 YEPD plates
2.
The plates were exposed to UV light in
groups of three at 0, 15, 30, and 45
seconds
3.
The plates were incubated for three
days at 32 degrees Celsius
4.
The colonies were counted; each
colony was assumed to have arisen
from a single cell
0.1 mL of suspended (-) Lys cells were plated
on each of 12 complete (-) Lys plates
2.
The plates were exposed to UV light in
groups of three at 0, 15, 30, and 45 seconds
3.
The plates were incubated for three days at
32 degrees Celsius
4.
Regular Yeast
The colonies were counted; each colony was
assumed to have arisen from a single cell
37.5
UV Exposure (-) Lys Yeast
Results
P-value:
0.000132
Colonies
30
22.5
15
7.5
0
0s
15s
30s
Seconds
45s
Dunnett's Tests
T-Crit: 3.75
Test
T-value
Interpretation
0 sec vs 15 sec
4.8805
Significant
0 sec vs 30 sec
2.1603
Insignificant
0 sec vs 45 sec
0.7945
Insignificant
UV Exposure (+) Lys Yeast
Results
112.5
P-value:
0.000454
90
Colonies
67.5
45
22.5
0
0
15
Seconds
30
45
Dunnett's Tests
T-Crit: 3.75
Test
T-value
Interpretation
0 sec vs 15 sec
1.87356
Insignificant
0 sec vs 30 sec
5.245857
Significant
0 sec vs 45 sec
6.958826
Significant
Effects of Selenium on UV stressed
Yeast's Mutagenesis Rate
40
Colonies
Blue:0%
Green: 0.001%
Yellow: 0.1%
0.001% Pvalue: 0.076
0.1% P-value:
0.330
30
20
10
0
0s
10s
UV Exposure Time
20s
Conclusion
•
Null Hypothesis can be rejected, as statistical
analysis did not show significant effect of selenium
on UV stressed yeast's mutagenesis rate
•
Alternative Hypothesis can be accepted, as
statistical analysis did show significant effect of UV
on both yeast strains in some experimental groups
•
It appears that in the concentrations tested,
Selenium is an ineffective anti-mutagen
Limitations
•
The amount of cells per plate may have varied
slightly
•
Plating was not perfectly synchronized
•
Slightly different positions in the UV hood
•
Inability to account for colony deaths due to
exposure to UV light
Extensions
•
Larger concentrations
•
Trypan Blue Assay to account for cell deaths
•
Use a different stressor
•
Use a different model
Sources
•
http://www.ncbi.nlm.nih.gov/pubmed/17588734
•
http://bioweb.uwlax.edu/bio203/s2007/nelson_andr/
•
http://missionscience.nasa.gov/ems/10_ultravioletwa
ves.html
•
http://users.rcn.com/jkimball.ma.ultranet/BiologyPag
es/A/AmesTest.html
ANOVA Example