Genetic Analysis of Carbonyl Reductase Function in Yeast

By Joshua BaumgartMentor: 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”

NADPH

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 mediumDefined medium missing

histadine

Defined medium missing

methionine

Direct genotyping by PCR

1 2 3 654 7 108 9 11

Template

Primers

△7:K

AN (p

os c

ontr

ol)

△1,

2:HI

S 7:

KAN

△

△8:K

AN (n

eg c

ontr

ol)

CBR7/KAN

△8:K

AN (p

os c

ontr

ol)

△1,2

:HIS

8:

KAN

△

△7:K

AN (n

eg c

ontr

ol)

△1,

2:HI

S (p

os c

ontr

ol)

△1,2

:HIS

7:

KAN

△

△8:K

AN (n

eg c

ontr

ol)

△1,2

:HIS

8:

KAN

△

CBR8/KAN CBR2/HIS

Expected band (bp) 723 723 - 462 - 865 -462 865 865

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

KAN

LEU2

LEU2

Direct genotyping by PCR

Template

Primers

△1,

2:HI

S 3:

LEU

#1

△

△1,

2:HI

S 3:

LEU

#4

△ △1,

2:HI

S 3:

LEU

#5

△

CBR2/LEU

△1,

2:HI

S 3:

LEU

#6

△ △1,

2:HI

S 3:

LEU

#7

△

△3:K

AN (n

eg c

ontr

ol)

△1,

2:HI

S 3:

LEU

#8

△

△1,

2:HI

S 3:

LEU

#9

△Expected band (bp) 1kb

1 2 4 53

△1,

2:HI

S 3:

LEU

#2

△ △1,

2:HI

S 3:

LEU

#3

△

1kb1kb1kb 1kb 1kb 1kb 1kb 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