Genetic Analysis of Carbonyl Reductase Function in Yeast

1. Genetic Analysis of Carbonyl Reductase Function in Yeast By Joshua Baumgart Mentor: Dr. Gary Merrill

2. Carbonyl reductase • Carbonyl reductase is an enzyme that reduces carbonyls (aldehydes and ketones) to their corresponding alcohols • The reaction requires a reducing agent called NADPH (NADPH is produced in all cells and represents “reducing power” NADPH NADPH

3. Relevance • Accumulation of carbonyl-containing compounds is potentially toxic to cells • Sources of carbonyl-containing compounds include: • External agents such as cigarette smoke, pollution, and automobile exhaust (which can lead to cancer) • Internal agents such as lipid breakdown products and intermediary metabolites

4. Saccharomycescerevisiae • Advantages of yeast as an experimental system • Grows rapidly (1.8 hour doubling time) • Can be maintained as haploid or diploid • Easy to delete, add, or replace genes • Genome completely sequenced (6022 genes) • Gene deletion project (about 1500 genes are essential) • Yeast contain ten genes with sequence similarity to mammalian carbonyl reductase • Individual deletion of any one of the ten yeast genes does not result in lethality

5. Ten yeast carbonyl reductase (CBR) genes

6. Library genotype • The library version of the genes obtained through the Saccharomyces Genome Database has the following genotype: Mat-α ura3 leu2 lys2 his3 MET15 yfg:KAN • The mutant that we used in the mating with the library to achieve a triple mutants was obtained through work done by Sarah Kerrigan summer research 2012 with the following genotype: Mat-a ura3 leu2 lys2 his3 met15 △cbr1/△cbr2:HIS3

7. Diploid genotype • During Winter and Spring term 2013, Merrill’s lab mated the remaining eight △cbr genes to the △cbr1/ △cbr2 double mutant created by Sarah Kerrigan creating the following genotype: Mat-a △cbr1 △cbr2:HIS3 CBR3 Mat-α CBR1 CBR2 △cbr3:KAN

8. Random spore analysis Defined medium missing histadine Rich medium Defined medium with kanamycin Defined medium missing methionine

9. Direct genotyping by PCR CBR8/KAN CBR2/HIS Primers CBR7/KAN △1,2:HIS △7:KAN △1,2:HIS △8:KAN △ 1,2:HIS (pos control) △8:KAN (neg control) Template △7:KAN (neg control) △8:KAN (neg control) △8:KAN (pos control) △7:KAN (pos control) △1,2:HIS △8:KAN △1,2:HIS △7:KAN 1 2 3 4 5 6 7 8 9 10 11 Expected band (bp) 723 723 - 462 462 - 865 865 865 -

10. Triple mutant genotype • From the random spore analysis I determined that a triple mutant missing △cbr1, △cbr2, and △cbr3 does not result in lethality created by the following genotype: • Merrill lab proved that a triple mutant created by the cross from Sarah Kerrigan’s double mutant and any one of the eight library mutants will not produce a lethality Mat-a △cbr1/△cbr2:HIS3 △cbr3:KAN

11. Summer project • Triple mutants lacking △cbr1, △cbr2, and one of the other eight Cbr genes were all viable • Create quadruple mutants missing △cbr1, △cbr2, △cbr3, and one of the other seven Cbr genes • Determine whether any of the quadruple mutants are inviable (produce synthetic lethality)

12. Approach 1. Replace △cbr3:KAN genewith △cbr3:LEU2 gene Mat-a △cbr1/△cbr2:HIS3 △cbr3:KAN Mat-a △cbr1/△cbr2:HIS3 △cbr3:LEU2 2. Make diploid by mating new mat-a triple mutant to mat-α library mutants mat-a △cbr1/△cbr2:HIS3 △cbr3:LEU2 CBR4 mat-αCBR1 CBR2 CBR3 △cbr4:KAN 3. Sporulate diploid, isolate random segregates, determine whether quadruple mutant is viable

13. 1. Replacing △cbr3:KAN genewith △cbr3:LEU2 gene • Prepared a LEU2 marker with KAN flanking sequences by PCR KAN5’ LEU2 KAN3’ pRS305 KAN5’ KAN3’ LEU2 • Transformed △cbr1,2:HIS3 △cbr3:KAN strainwith LEU2 fragment • Selected transformants on medium lacking leucine

14. CBR3 KAN KAN LEU2 LEU2

15. Direct genotyping by PCR Primers CBR2/LEU △ 1,2:HIS △3:LEU #9 △ 1,2:HIS △3:LEU #8 Template 1 2 3 4 5 △ 1,2:HIS △3:LEU #7 △3:KAN (neg control) △ 1,2:HIS △3:LEU #5 △ 1,2:HIS △3:LEU #6 △ 1,2:HIS △3:LEU #1 △ 1,2:HIS △3:LEU #2 △ 1,2:HIS △3:LEU #3 △ 1,2:HIS △3:LEU #4 Expected band (bp) 1kb 1kb 1kb 1kb 1kb 1kb 1kb 1kb 1kb -

16. 2. Make diploid • After confirming transformation maker conversion, I mated triple mutant to each of the seven remaining △cbr:KANsingle mutants • For example, mating to △cbr4:KAN is expected to give a diploid with the following genotype: Mat-a △cbr1/△cbr2:HIS3 △cbr3:LEU2 CBR4 Mat-α CBR1 CBR2 CBR3 △cbr4:KAN

17. 3. Sporulate diploid • Transferred diploid to nutrient-deficient plates to induce sporulation • Isolated spores by ether treatment • Plated spores at low dilution on rich medium to induce germination • Picked random colonies to micro-titer wells • Replicaplated micro-titer dish to selective conditions

18. Random spore analysis Rich medium Defined medium with kanamycin Defined medium missing Leucine Defined medium missing histadine

19. Summary • I converted the△cbr1,2:HIS3 △cbr3:KAN triple mutant to a △cbr1,2:HIS3 △cbr3:LEU2 triple mutant • I mated the new triple mutant to seven single mutants to derive diploids • I analyzed the diploids by random spore analysis and determined that all seven quadruple mutants were viable (no synthetic lethality) • I confirmed the genotype of all derived strains by PCR • The genotype of the quadruple mutant is: Mat-a △cbr1/△cbr2:HIS3 △cbr3:LEU2 △cbr:KAN

20. Results • △cbr4 and △cbr5 is a confirmed transformant for the LEU2 gene integration • I have successfully moved △cbr6-8 to the transformation step.

21. Future research direction • Continue the process of homologously integrating the LEU2 gene into △cbr6-8 to verify that any quadruple mutant made by the other Cbr genes would not result in lethality. • If no quadruple mutants show synthetic lethality, knockout a fifth gene creating a quintuple mutant to see if any new combination would result in a lethality.

22. Acknowledgments • Dr. Gary Merrill • Ray, Frances, and Dale Cripps Scholarship fund • Dr. Kevin Ahern • Oregon State University Undergraduate Summer Research Program • Merrill lab • Jason Mah • Thi Nguyen