Kris Sabatini Selenium Effects on UV Stressed Yeast’s Mutagenesis Rate CCHS, Grade 10 3 rd 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, photo- aging, nausea, and possibly skin cancer. - Shorter non-lethal exposure times can lead to mutations.

Electro-magnetic spectrum

Selenium -Atomic Number,34 Atomic Weight, 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 AcCoA CoA HC Synthase Homocirate LYS7 Water Homoaconitate Homoisocitrate a - Ketoadipate a- Aminoadipate a- Aminodipate Semialdehye SaccharopinaLysine LYS4 LYS12 aAA- Aminotranfease LYS2 LYS9 LYS1 NAD NADH CO 2 Glutamate a-Ketoglutrate ATP PP NADPH NADP Glutamate NADPH NADP Water NADP; NADP a- Ketoglutarate Lysine’s 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 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.

Was there a significant interact between the variables of Selenium supplementation and UV Light cell mutagenesis? Did the Selenium affect the mutagenesis rate of non- stressed 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? Important Questions

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 Materials

Procedure 1. A strain of yeast (-) Lys phenotype was grown for 2 days in YEPD media 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 SDFSelenium Stock YeastTotal Volume Tube 1 (0x)1 mL0 mL1 mL2 mL Tube 2 (1x).99 mL.01 mL1 mL2 mL Tube 3 (10x).9 mL.1 mL1 mL2 mL

7. The cells were allowed to sit for 15 min 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 ) 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 32 o C. 11. The colonies were counted and recorded. Each colony assumed to have arisen from 1 cell.

Results

ANOVA Analysis Was there a significant interact between the variables of Selenium supplementation and UV Light in cell mutagenesis? P-value E-17, significant Did the Selenium affect the mutagenesis rate of non-stressed yeast? P-value E-13, significant Did the UV light affect the mutagenesis rate of yeast without selenium added? P-value E-13, significant Did the selenium affect the mutagenesis rate of 10 second exposure group? P-value , insignificant Did the selenium affect the mutagenesis rate of the 20 second exposure group? P-value , significant

Dunnett’s Test Variable ComparedT-valueVariation, 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 0[x] 20s 10[x] 10s compared to 0[x] 20s T-value =.192 T-value = 8.48 Insignificant Significant T crit = 4.03

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