Role of Clock Gene period in Starvation Resistance

A Research Project by: Sarah StrawnMentored by: Dr. Jaga Giebultowicz

Drosophila melanogaster

Circadian Rhythms

24 hour Cycles that keep you on schedule.

Circa- about/aroundDia- day

Each cycle influences aspects of your body’s function. -Sleep/wake cycles, heart rate, blood pressure, and hormone secretion.

There are more than 100Circadian rhythms in the human body.

Period Gene and Emergence

Genetic Basis of Biological Clock -Mutants that lack functional period are arrhythmic.

Period (per) was the first clock gene discovered based on a mutationin 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 3 4 days

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Why Flies?

Per gene is conserved between flies and mammals (including humans)One per gene in Drosophila (but: Per1, Per2, Per3 in mice)

Drosophila is an established model for biomedical research

More than 75% of human disease loci have counterparts in fly.

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

HypothesisThe 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.

Mutant per- null flies

Survival under starvation

w Control Flies normal level of per

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.

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.

ResultsStarvation Resistance in Day 15 Females

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w wper0Genotype

Mean Survival Time

(hrs)VIRGINS

MATED

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!

Do mutants weigh more than control flies at the onset of starvation?

Food Consumption

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control mutant

Geneotype

Absorbance (625nm)

Average Mass of Flies

Results: Both controls and mutants start out on equal footing.

It was determined that average mass was not statistically significant.

Mass of Mated Females Day 15

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w wper0

Geneotype

Average Mass in (mg)

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.

I

These results suggest that mutants have problems with metabolism and storage of nutrients What metabolic advantage do control flies have over 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 ProbeMutant

Fat cells around sperm storage

organ

Fat cells near alimentary

tract

Fat granuleFat granule

Preparations stained with: Hoechst DNA stain, Lysotracker and BODIPY neutral fat probe

Control

Research in progress

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

Special Thanks

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

»Jaga Giebultowicz