marisa mcallister dr. jaga giebultowicz lab oxidative stress response genes, cncc, keap1, and gstd1:...
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Marisa McAllister
Dr. Jaga Giebultowicz Lab
Oxidative Stress Response Genes, cncc, keap1, and gstd1: A
Circadian Connection?
Circadian Rhythms in Daily LifeControls rhythms in daily life functionsRhythms controlled by genesInternal clock disrupted
during travel = jetlagevening or rotating work shifts (15%)
Associated with insomnia, diabetes, depression, heart attacks, and cancer
Working hypothesis: controls the repair of oxidative stress
Oxidative stress has been implicated as a cause of aging
-Order: Diptera-Commonly called a fruit fly-Ideal model organism for studying genetics
-Canton Strain (CS) is the wild type control-Negative feedback loop: period (per) and timeless (tim) -Positive feedback loop: clock (clk) and cycle (cyc)
-per01 is the mutant loss-of-function gene-The time of day at which flies were exposed to H2O2 affected how well they were able to repair damage- per01 mutants are more susceptible to H2O2 than CSp flies at 9am and 5am-H2O2 levels in wild-type flies were higher during the day than at night
-Nrf2 = transcription factor in the leucine zipper family-Keap1 = actin binding protein that is Nrf2’s repressor
-A homolog of Nrf2 has been found in D. melanogaster-The gene cncC has a similar location, function, and N terminus to Nrf2-gstD1 is an oxidative stress response gene that cncC activates
Circadian genes
Transcription
Oxidative Repair Mechanisms (gstd1)
?
-Clock genes regulate the response to oxidative stress by stimulating expression of cncC/keap1.-Prediction: expression levels of keap1, cncC and gstd1 will cycle in a 24 hour period.-Prediction: expression of keap1 and cncC, will be reduced in the per01 or cyc01 mutants.-Prediction: gstD1 should be down-regulated in the mutants.
-Collect 5-day old male CSp, per01, CSc and cyc01 flies every 4 hours for 24 hours-Separate heads and bodies-Run quantitative real time polymerase chain reaction
gstd1 mRNA Expression Levels
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
2.0Control
per01
Hours after lights on
Rel
ativ
e E
xpre
ssio
n (
RQ
)
- gstd1 mRNA is cycling with the circadian day.- gstd1 mRNA is cycling with the circadian day.
- May not be controlled by - May not be controlled by periodperiod..
cncC mRNA Expression Levels
- cncC does not appear to cycle in the circadian day.
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
2.0Control
per01
Hours after lights on
Rel
ativ
e E
xpre
ssio
n (
RQ
)
keap1 mRNA Expression Levels
-
0 4 8 12 16 20 24
0
1
2
3Control
per01
Hours after lights on
Rel
ativ
e E
xpre
ssio
n (
RQ
)
- keap1 does not appear to cycle in the circadian day.
gstd1 mRNA Expression Levels
0 4 8 12 16 20 24
0.0
0.5
1.0
1.5
2.0
2.5Control
Cyc01
Hours after lights on
Rel
ativ
e E
xpre
ssio
n (
RQ
)
- gstd1 appears to cycle in the control flies, but not in the cyc01 flies
cncC mRNA Expression Levels
0 4 8 12 16 20 24
0
1
2
3Control
Cyc01
Hours after lights on
Rel
ativ
e E
xpre
ssio
n (
RQ
)
- cncC is expressed at a higher level in the control flies than in the cyc01 flies.
keap1 mRNA Expression Levels
0 4 8 12 16 20 24
0
1
2
3
4Control
Cyc01
Hours after lights on
Rel
ativ
e E
xpre
ssio
n (
RQ
)
- keap1 is expressed at a higher level in the control flies than in the cyc01 flies.
Circadian Rhythms and LightIf a gene is controlled by the circadian clock it should be:
Rhythmic in light-dark conditionsRhythmic in constant darknessArrhythmic in constant light
gstd1 mRNA expression levels in differing light conditions
8 20 8 20 8 200.0
0.5
1.0
1.5Light-DarkConstant DarknessConstant Light
Hours after lights on
Rel
ativ
e E
xpre
ssio
n (
RQ
)
-gstd1 appears to be a rhythmic gene because it is cycling in LD and DD, but not cycling in LL
Summary/ConclusionsSpecific findings:keap1 and cncC do not appear to cycle in a 24 hour period. Expression of keap1 and cncC are reduced in cyc01 mutants
compared to controls.Expression of keap1 and cncC are similar in per01 mutants
compared to controls.gstd1 appears to cycle in a circadian day with a peak at 5pm
and a trough at 5am.
Conclusion:-Unclear whether clock genes regulate the response to oxidative stress through the cncC/keap1 pathway.-gstd1 may be regulated by the circadian clock independently of cncC/keap1.
AcknowledgementsJaga GiebultowiczLaura BeaverGiebultowicz Lab MembersKevin AhernUniversity Honors CollegeHHMIURISC