Transcript Genetic Analysis of Carbonyl Reductase Function in Yeast By Joshua Baumgart

Genetic Analysis of
Carbonyl Reductase Function in Yeast
By Joshua Baumgart
Mentor: Dr. Gary Merrill
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
Relevance
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
Accumulation of carbonyl-containing compounds is
potentially toxic to cells
Sources of carbonyl-containing compounds include:
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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)
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Yeast contain ten genes with sequence similarity to
mammalian carbonyl reductase
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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
△yir036c:Kan
△ykr009c:Kan
△ykl071w:Kan
△yor246c:Kan
△ydl114w:Kan
△yil124w:Kan
△ymr226c:Kan
△ylr426w:kan
△ykl055c:Kan
△cbr1
△cbr 2
△cbr 3
△cbr 4
△cbr 5
△cbr 6
△cbr 7
△cbr 8
△cbr 9
△cbr 10
Library genotype
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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
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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
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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
Rich medium
Defined
medium
with
kanamycin
Defined
medium
missing
histadine
Defined
medium
missing
methionine
Expected band (bp)
5
6
723 723
462
△8:KAN (neg control)
4
△1,2:HIS △8:KAN
3
△1,2:HIS △7:KAN
2
△ 1,2:HIS (pos control)
△1,2:HIS △8:KAN
CBR7/KAN
△7:KAN (neg control)
△8:KAN (pos control)
1
△8:KAN (neg control)
Template
△1,2:HIS △7:KAN
Primers
△7:KAN (pos control)
Direct genotyping by PCR
CBR8/KAN
462 -
CBR2/HIS
7
8
9
10
11
865 865 865 -
Triple mutant genotype
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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:
Mat-a △cbr1/△cbr2:HIS3 △cbr3:KAN
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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
Summer project
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Triple mutants lacking △cbr1, △cbr2, and one of the other
eight Cbr genes were all viable
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Create quadruple mutants missing △cbr1, △cbr2, △cbr3,
and one of the other seven Cbr genes
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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: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
mat-α
CBR1
CBR2
CBR3
CBR4
△cbr4:KAN
3. Sporulate diploid, isolate random segregates, determine whether
quadruple mutant is viable
1. Replacing △cbr3:KAN gene with △cbr3:LEU2 gene
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Prepared a LEU2 marker with KAN flanking sequences by PCR
LEU2
pRS305
KAN5’
LEU2
KAN3’
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Transformed △cbr1,2:HIS3 △cbr3:KAN strain with LEU2 fragment
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Selected transformants on medium lacking leucine
CBR3
KAN
KAN
LEU2
LEU2
Template
Expected band (bp)
1
△ 1,2:HIS △3:LEU #8
3
4
5
1kb 1kb 1kb 1kb 1kb 1kb 1kb 1kb 1kb
△3:KAN (neg control)
△ 1,2:HIS △3:LEU #9
△ 1,2:HIS △3:LEU #7
△ 1,2:HIS △3:LEU #5
△ 1,2:HIS △3:LEU #4
△ 1,2:HIS △3:LEU #3
Primers
△ 1,2:HIS △3:LEU #6
2
△ 1,2:HIS △3:LEU #2
△ 1,2:HIS △3:LEU #1
Direct genotyping by PCR
CBR2/LEU
-
2. Make diploid
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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
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Transferred diploid to
nutrient-deficient plates to
induce sporulation
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Isolated spores by ether
treatment
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Plated spores at low dilution
on rich medium to induce
germination
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Picked random colonies to micro-titer wells
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Replicaplated micro-titer dish to selective conditions
Random spore analysis
Rich
medium
Defined
medium
missing
histadine
Defined
medium
with kanamycin
Defined
medium
missing
Leucine
Summary
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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
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△cbr4 and △cbr5 is a confirmed transformant for the
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LEU2 gene integration
I have successfully moved △cbr6-8 to the transformation
step.
Future research direction
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
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
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Dr. Gary Merrill
Ray, Frances, and Dale Cripps Scholarship fund
Dr. Kevin Ahern
Oregon State University Undergraduate Summer
Research Program
Merrill lab
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Jason Mah
Thi Nguyen