Transcript Role of Clock Gene period

Role of Clock Gene period in
Starvation Resistance
Drosophila melanogaster
A Research Project by: Sarah Strawn
Mentored by: Dr. Jaga Giebultowicz
Circadian Rhythms
24 hour Cycles that
keep you on schedule.
Circa- about/around
Dia- day
Each cycle influences
aspects of your body’s
function.
-Sleep/wake cycles, heart
rate, blood pressure, and
hormone secretion.
There are more than 100
Circadian rhythms in the human body.
Period Gene and Emergence
No. of flies emerged
Genetic Basis of
Biological Clock
-Mutants that lack
functional period
are arrhythmic.
Period (per) was the first
clock gene discovered
based on a mutation
in emergence patterns.
Period was so named
due to other alleles that
produced emergence rhythm
with periods either shorter or
longer than 24 hours.
1
2
days
3
4
Why Flies?
Drosophila is an established model for
biomedical research
More than 75% of human disease
loci have counterparts in fly.
Per gene is conserved between flies and mammals (including humans)
One per gene in Drosophila (but: Per1, Per2, Per3 in mice)
The period gene is the focus of this
study
• Period is expressed in most cells of the body
• Loss of per function produces many phenotypes:
– Behavioral arrhythmia
– Disruption of courtship song
– Decrease in fitness
• may be involved in the regulation of metabolism
– Starvation Resistance is an indirect measure of metabolism
Hypothesis
The period gene is involved in the regulation of
starvation resistance in flies.
If the hypothesis is correct, the dosage of period gene
should be a factor in determining how well flies respond to
starvation.
Prediction: Flies with mutated period gene that produce no period
protein will have the shorter survival time than flies with active
period gene and normal levels of period protein.
Survival under starvation
w Control Flies
normal level of per
Mutant per- null flies
Objectives
- Examine possible links between levels of
period gene expression and starvation
resistance.
- Identify mechanism by which period
affects survival under starvation.
Genotypes of flies used in this study
• w
w per+/w per+;; – control flies that carry a
white marker and normal period gene
• w per0
w per0/w per0;; – containing inactive period
gene (per- null) mutants.
Note: all flies were isogenized meaning that their genetic
background is similar except for the genes indicated
(per, w)
Starvation test
Flies are placed into
vials with filter paper
and water. Deaths are
recorded and water
replaced every 12 hours.
Results
Starvation Resistance in Day 15 Females
160
Mean Survival Time
(hrs)
140
120
100
VIRGINS
MATED
80
60
40
20
0
w
wper0
Genotype
Resistance to starvation is significantly lower in mutants than in control females
This effect is not evident in virgins but is pronounced in mated females
>>We concentrated on mated females in further studies
Do control flies eat more than mutants?
How can we measure the amount
of food flies consume?
Blue diet allows us to measure
the amount of food flies eat.
When a fly ingests food the
alimentary canal is stained
blue.
Concentrations of blue dye in the gut
were measured using spectrometer
Mutant flies eat more than control
flies!
Food Consumption
0.2
0.18
Absorbance (625nm)
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
control
mutant
Geneotype
Do mutants weigh more than control flies at the onset of starvation?
Average Mass of Flies
Mass of Mated Females Day 15
1.4
Average Mass in (mg)
1.2
1
0.8
0.6
0.4
0.2
0
w
wper0
Geneotype
Results: Both controls and mutants start out on equal footing.
It was determined that average mass was not statistically significant.
Summary of Results
• Control flies live longer than mutants when
subjected to starvation.
• Mutants consume more food than control.
• Both control and mutants have approximately
equal mass before starvation.
These results suggest that mutants have problems with
metabolism and storage of nutrients
What metabolic advantage do control flies have over
I
mutants?
Are they better able to make use of their resources when starved?
Do they have more fat reserves?
To survive starvation, cells use
autophagy
• During autophagy proteins that can be spared are
digested in lysosomes and provide “recycled” nutrients
• Lysotracker is a pH sensitive probe that can detect
autophagy in larval fat body
Lysotracker turns red under
acidic pH of active lysosomes
and Hoechst stains nucleus
blue.
This dye was used to compare
the intensity of autophagy
between starved control and
mutant flies.
Fat Content with Green Probe
Control
Mutant
Fat cells around
sperm storage
organ
Fat cells near
alimentary
tract
Fat granule
Preparations stained with: Hoechst DNA stain, Lysotracker and BODIPY neutral fat probe
Fat granule
Research in progress
• Measure triglyceride levels at the onset
and during the course of starvation in
control and mutant flies.
Special Thanks
•
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Howard Hughes Medical Institute
URISC
Kevin Ahern
Brandy Rush
Channa Newell
Jessica Bruer
Andy Frazer
»Jaga Giebultowicz