mineral oil effects on yeast mutagenesis luke giannetta second year in pjas central catholic high...
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MINERAL OIL EFFECTS ON
YEAST MUTAGENESIS
Luke GiannettaSecond Year in PJAS
Central Catholic High School10th Grade
Unrefined mineral oil is a known carcinogen(American Cancer Society) Studies by:
International Agency for Research on CancerNational Toxicology Program
Principal Ingredients D-alpha tocopherol (Vitamin E) Liquid Paraffin
MINERAL OIL
Saccharomyces cerevisiae Commonly used model Tolerant and safe to culture Has similar reproduction,
metabolism, and chemistryas other more advanced eukaryotic cells
(-) Lys Special strain unable to produce lysine
CELL MODEL
Lysine codons AAA, AAG
(-) lysine yeast mutantsused in research
Lys 2 mutants are missingan enzyme functionwithin the lysine biosynthesis pathway
Result: Cells require lysine supplementation
LYSINE
Developed by Bruce Ames (c. 1970)
Tests for mutagenic/anti-mutagenic properties of various chemicals
Used a (-)-histidine mutant Salmonella(single-point substitution)Bacteria cannot synthesize histidine due to mutation
Exposure to suspected mutagen correlated with increased reversion (mutation) rate
Visible colonies appearing on complete (-)Histidine media evidence of mutation through reversion
Obviously, a lower limit on mutation
Assayed only 1 DNA site in genome
AMES TEST
MODIFIEDAMES TEST
The number of reverted colonies of yeastcan be correlated with the rate of
mutation.
A reversion at that pointcan result in a reversion
back to wild type yeast (lys +)
Short-wavelength electromagnetic waves
greater energy than visible light
wavelengths range from 400nm to 10nm
Sun’s UV rays absorbed by ozone layer
Causes direct DNA damage (mutagen)
ULTRAVIOLET (UV) RAYS
PURPOSE To determine the effects of mineral oil
(suspected mutagen) on the mutagenesis rateof (-) Lys yeast
To determine the effects of UV light (known mutagen) on the mutagenesis of (-) Lys and the survivorship of wild-type yeast
HYPOTHESES Null Hypothesis
Mineral oil will not have a significant effect on the mutagenesis rate of yeast.
Alternate HypothesisMineral oil will have a significant effect onthe mutagenesis rate of yeast.
(-) Lysine agar plates1% yeast nitrogen basew/o amino acids2% glucose1 mM amino acid mix1.5% agar
YEPD plates UV light hood
(LD-50 on yeast is 30 s) Sterile dilution fluid (SDF)
10 mM KH2PO4, 1 mM MgSO4
1 mM CaCl2, 100 mM NaCl
SDF Test Tubes Sterile pipette tips,
microplates
Vortex Side-arm flask Spreader bar Ethanol Micro burner (-) Lysine Saccharomyces
cerevisiae Wild type (+) Lys
Saccharomyces cerevisae Rubber gloves Test tubes Microtubes Test tube rack Rite Aid ® Mineral Oil
MATERIALS
1. Strain of yeast (-) Lys phenotype grown for 2 days in YEPD media.
2. Sterile yeast pellet washed with SDF to remove any residual nutrients (lysine)
3. Stock re-suspended and stored in com. (-) Lys mediafor 2 days
4. A 100% stock and a 10% sub-stock of the mineral oil were made by diluting the variable with sterile water.
5. The pellet in SDF was re-suspended.
PROCEDURE
6. The following ingredients were pipetted into sterile microtubules
PROCEDURE (CONT’D)
Water Variable YeastTotal
Volume
0% 0.8 mL 0 mL 0.2 mL 1 mL
0.1% 0.7 mL0.1 mL(of 10%
substock)0.2 mL 1 mL
1% 0.79 mL 0.01 mL 0.2 mL 1 mL
10% 0.7 mL 0.1 mL 0.2 mL 1 mL
7. Cells were allowed to sit for 25 mins.
8. 0.1 mL of each tube plated onto 6 complete (-) Lys plates(necessary to show cells that have reverted via mutation to wild-type (+) Lys)
9. Remaining 0.4 mL of each tube was split into two 0.2 mL aliquots and plated onto 2 complete (-) Lys plates
10. Plates were incubated for 5 days at 32 °C
11. Colonies counted and recorded.Each colony assumed to have arisen from a single cell.
PROCEDURE (CONT’D)
(-) Lys Yeast
0.1 mL of suspended (-) Lys cells were plated on each of 12 complete (-) Lys plates
Plates exposed to UV light in groups of three – at 0, 15, 30, and 45 seconds
Plates were incubated for three days at 32 °Celsius
Colonies were counted, each colony assumbed to have arisen from a single cell
Regular Yeast
0.1 mL of suspended Saccharomyces ceravisae cells were plated on each of 12 YEPD plates
Plates were exposed to UV light in groups of three at 0, 15, 30, and 45 seconds
Plates incubated for three days at32 °Celsius
Colonies were counted, each colony assumed to have arisen from a single cell
UV EXPOSURE PROCEDURE
0 sec 15 sec 30 sec 45 sec0.0
7.5
15.0
22.5
30.0
37.5
(-) LYS UV EXPOSURE RESULTS#
of
Colo
nie
s
UV Light Effects on (-) Lys Yeast
P value = 0.000132
Exposure Time (s)
Test T Value Interpretation
0 sec vs. 15 sec 4.8805 Significant
0 sec vs. 30 sec 2.6103 Not Significant
0 sec vs. 45 sec 0.7945 Not Significant
DUNNETT’S TEST RESULTS
T-Critical = 3.75
P-value = 0.000454
REGULAR YEAST UV EXPOSURE RESULTS
# o
f C
olo
nie
s
UV Light Effects on Yeast Survivorship
0 15 30 4530
40
50
60
70
80
90
P value = 0.000454
Exposure Time (s)
Test T Value Interpretation
0 sec vs. 15 sec 1.87356 Not Significant
0 sec vs. 30 sec 5.245857 Significant
0 sec vs. 45 sec 6.958826 Significant
DUNNETT’S TEST RESULTS
T-Critical = 3.75
0.0% 0.1% 1.0% 10.0%0
1
2
3
4
MINERAL OIL EXPOSURE RESULTS
Mineral Oil Effect on Mutagenesis
# o
f C
olo
nie
s
Mineral Oil Concentration
P Value = 0.032446
Test T Value Interpretation
0% vs. 0.1% 0.21934 Not Significant
0% vs. 1%sec 1.97406 Not Significant
0% vs. 10% 2.63208 Not Significant
DUNNETT’S TEST RESULTS
T-Critical = 2.88
None of the concentrations of mineral oil showed ability to significantly affect the rate of yeast mutagenesis
Null Hypothesis was accepted Alternate Hypothesis was rejected
CONCLUSIONS
Limitations
Slightly out-of-synced plating which leads to slightly different exposure times to mineral oil
Inability to control the exact amount of cells on each plate (minor difference overshadow by massive amount of cells)
Slight positioning differences in UV Oven Inability to account for cell deaths due UV Light
LIMITATIONS AND EXTENSIONS
Extensions
Different model
Reduce lag time with lab assistants
Trypan Blue Assay to account for cell deaths
A future experiment testing mineral oil's effects on mammalian and cancerous cell lines to see if it promotes uncontrollable growth.
LIMITATIONS AND EXTENSIONS
http://www.britannica.com/EBchecked/topic/442584/paraffin-hydrocarbon
https://shop.riteaid.com/rite-aid-pharmacy-mineral-oil-usp-16-fl-oz-1-pt-473-ml-0033067
http://www.cancer.org/cancer/cancercauses/othercarcinogens/generalinformationaboutcarcinogens/known-and-probable-human-carcinogens
http://www.aacr.org/home/public--media/aacr-in-the-news.aspx?d=2740
http://dontdip.tamu.edu/ingredients.htm
http://www.sciencemag.org/content/274/5286/430.abstract?ijkey=7dd94e096ea549bac90bce0ec51acb6422cbb1a4&keytype2=tf_ipsecsha
http://www.iarc.fr/en/media-centre/pr/2004/pr154.html
SOURCES
ANOVA
Seconds Exposed 0 15 30 45 Wild Type Yeast
91 74 69 49
80 74 46 45
92 80 50 39
Anova: Single Factor
SUMMARY
Groups Count Sum Average Variance
Column 1 3 263 87.66667 44.33333
Column 2 3 228 76 12
Column 3 3 165 55 151
Column 4 3 133 44.33333 25.33333
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 3478.917 3 1159.639 19.93649 0.000454 4.066180551
Within Groups 465.3333 8 58.16667
Total 3944.25 11