Role of Clock Gene period in Starvation Resistance

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Role of Clock Gene period in Starvation
Resistance
Drosophila melanogaster

• A Research Project by Sarah Strawn
• Mentored by Dr. Jaga Giebultowicz

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Circadian Rhythms
24 hour Cycles that keep you on
schedule. Circa- about/around Dia- day Each
cycle influences aspects of your bodys
function. -Sleep/wake cycles, heart rate, blood
pressure, and hormone secretion.
There are more than 100 Circadian rhythms in the
human body.
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Period Gene and Emergence
Period (per) was the first clock gene discovered
based on a mutation in emergence patterns.
Genetic Basis of Biological Clock -Mutants
that lack functional period are arrhythmic.
Period was so named due to other alleles that
produced emergence rhythm with periods either
shorter or longer than 24 hours.
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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)
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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

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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
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Objectives

• Examine possible links between levels of period
  gene expression and starvation resistance.
• Identify mechanism by which period affects
  survival under starvation.

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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)

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Starvation test
Flies are placed into vials with filter paper
and water. Deaths are recorded and water
replaced every 12 hours.
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Results
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 gtgtWe concentrated
on mated females in further studies
Do control flies eat more than mutants?
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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
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Mutant flies eat more than control flies!
Do mutants weigh more than control flies at the
onset of starvation?
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Average Mass of Flies
Results Both controls and mutants start out on
equal footing.
It was determined that average mass was not
statistically significant.
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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
mutants?
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Are they better able to make use of their
resources when starved? Do they have more fat
reserves?
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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.
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Fat Content with Green Probe
Control
Mutant
Fat cells around sperm storage organ
Fat cells near alimentary tract
Preparations stained with Hoechst DNA stain,
Lysotracker and BODIPY neutral fat probe
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Research in progress

• Measure triglyceride levels at the onset and
  during the course of starvation in control and
  mutant flies.

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Special Thanks

• Howard Hughes Medical Institute
• URISC
• Kevin Ahern
• Brandy Rush
• Channa Newell
• Jessica Bruer
• Andy Frazer
• Jaga Giebultowicz