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

 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” Carbonyl reductase NADPH

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

Saccharomyces cerevisiae  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

Ten yeast carbonyl reductase (CBR) genes Gene knockouts (SGD nomenclature) My nomenclature △ yir035c:Kan △ cbr1 △ yir036c:Kan △ cbr 2 △ ykr009c:Kan △ cbr 3 △ ykl071w:Kan △ cbr 4 △ yor246c:Kan △ cbr 5 △ ydl114w:Kan △ cbr 6 △ yil124w:Kan △ cbr 7 △ ymr226c:Kan △ cbr 8 △ ylr426w:kan △ cbr 9 △ ykl055c:Kan △ cbr 10

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

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

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

Direct genotyping by PCR Template Primers △ 7:KAN (pos control) △ 1,2:HIS △ 7:KAN △ 8:KAN (neg control) CBR7/KAN △ 8:KAN (pos control) △ 1,2:HIS △ 8:KAN △ 7:KAN (neg control) △ 1,2:HIS (pos control) △ 1,2:HIS △ 7:KAN △ 8:KAN (neg control) △ 1,2:HIS △ 8:KAN CBR8/KANCBR2/HIS Expected band (bp)

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

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)

Approach 1. Replace △ cbr3:KAN gene with △ cbr3:LEU2 gene Mat-a △ cbr1/ △ cbr2:HIS3 △ cbr3:LEU2 Mat-a △ cbr1/ △ cbr2:HIS3 △ cbr3:KAN 2. Make diploid by mating new mat-a triple mutant to mat-α library mutants 3. Sporulate diploid, isolate random segregates, determine whether quadruple mutant is viable mat-a △ cbr1/ △ cbr2:HIS3 △ cbr3:LEU2 CBR4 mat-α CBR1 CBR2 CBR3 △ cbr4:KAN

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

CBR3 KAN LEU2

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

2. Make diploid  After confirming transformation maker conversion, I mated triple mutant to each of the seven remaining △ cbr:KAN single 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

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

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

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

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

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.

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