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Selenium Effects on UV Stressed Yeast’s Mutagenesis Rate Kris Sabatini CCHS, Grade 10 3 rd Year in PJAS
Ultraviolet Rays - Ultraviolet (UV) rays have shorter wavelengths than visible light. They range from 400 nm to 10 nm. - 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.
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
Lysine a - Ketoglutarate Ac. Co. A HC Synthase Homocirate Water Homoaconitate • Lysine’s codons are AAA and AAG NADH CO 2 Homoisocitrate 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 Glutamate NADPH NADP Water a - Ketoadipate a. AA- Aminotranfease a- Aminoadipate a- Aminodipate Semialdehye NADP; NADP a- Ketoglutarate Lysine LYS 2 LYS 9 Saccharopina LYS 1 LYS 4 LYS 12 a-Ketoglutrate ATP PP NADPH NADP LYS 7
Ames Test -Developed to test the mutagenic and anti-mutagenic properties of various chemicals by Bruce Ames in 1970 s. - 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] (10 m. M KH 2 PO 4, 10 m. M K 2 HPO 4, 1 m. M Mg. SO 4, . 1 m. M Ca. Cl 2, 100 m. M Na. Cl) • 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 m. L 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 m. L tubes. X = to the recommended diet intake 200 mcg/ 5 L SDF Selenium Stock Yeast Total Volume Tube 1 (0 x) 1 m. L 0 m. L 1 m. L 2 m. L Tube 2 (1 x) . 99 m. L . 01 m. L 2 m. L Tube 3 (10 x) . 9 m. L . 1 m. L 2 m. L
7. The cells were allowed to sit for 15 min. 8. . 1 m. L 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, 0 s, 10 s and 20 s. 10. The plates were allowed to incubate for 3 days at 32 o 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 0 x Selenium Dose 20 1 x Selenium Dose 15 10 x Selenium Dose 10 5 0 0 s 10 s Exposure Time to UV Light 20 s
ANOVA Analysis • Was there a significant interact between the variables of Selenium supplementation and UV Light in cell mutagenesis? • P-value - 3. 38 E-17, significant • Did the Selenium affect the mutagenesis rate of non-stressed yeast? • P-value - 4. 47 E-13 , significant • Did the UV light affect the mutagenesis rate of yeast without selenium added? • P-value - 8. 04 E-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] 0 s compared to control 10[x] 0 s compared to control T-value = 3. 23 T-value =. 230 Insignificant 1[x] 10 s compared to 0[x] 10 s 10[x]10 s compared to 0 x 10 s T-value =. 232 T-value = 2. 36 Insignificant 1[x] 10 s compared to T-value =. 192 0[x] 20 s T-value = 8. 48 10[x] 10 s compared to 0[x] 20 s 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 10 s group. The ANOVA analysis and Dunnett’s tests supports this. • The Selenium also appeared to have an effect on the 20 s group but only in the 10 x 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