Circadian Rhythms: Lecture 5 The Plot Thickens...

What makes the clock tick? Hands of the SCN “Main Oscillator” (SCN cells) But what about at the molecular level? GENES (contain the instructions that tells a cell what its job will be) Cells contain a newly discovered protein (clock protein) that regulates gene function and which shows 24-hr variations in cellular levels that appears to account for 24-hr variations in neuronal activity

Genes that underlie our Biological Clock How do reseachers study this? How do you begin studying genes 1.Simple Model 2.Short-life span (inherited mutations)

Drosophila (Fruit Fly) Ideal for Genetic Studies 1. Short Life Span: breed & interbreed 1,000s of generations 2. Size of fly is Small: Lab set up minimal! Genotype = gene Phenotype = outward behavior attributed to function of the gene

So…..We want to know if there is a particular gene (genotype) responsible for circadian rhythms (phenotype) Start backwards  we don’t know the gene but we do know the behavior  disruption in circadian rhythms We need to measure circadian rhythms in Drosophila = Active during the day = 12L/12D cycle

Measurement of Activity in the Fruit FLy

Get the phenotype = disruption in 12L/12D Cycle Mutant Genotype 1. Fed the flies mutagens 2. 2,000 of their progeny (200 th try) 3. Found 3 flies showing disrupted 12L/12D cycles..first to tie behavior to gene… (Period) 19 hr cycle No cycle 29 hr cycle Konopka & Benzer (1970)

16 years later Michael Young (1986) Found that 3 flies had alterations in a single gene X Chromosome Period: PER PER is somehow involved in -producing circadian rhythm - setting the length of the rhythm

Is this the only gene involved? Start from the drawing board Fed flies mutagen 7,000 Progeny ONE Fly!..had no circadian rhythm Mutation on Chromosome 2 Named it TIMELESS = TIM…. So do these two genes work together?

Cell body DNA nucleus Genes are made of DNA- located on Chromosome DNA: instructions for making proteins (A=T G=C) DNA never leaves the cell Instructions are copied onto RNA – mRNA (transcription) mRNA: leaves nucleus enters the cytoplasm with the molecular recipe for amino acids Rough ER (ribosomes) – translation amino acids proteins mRNA ER-ribosomes PER/TIM Proteins

Do PER & TIM work together? So, isolated both PER & TIM (genes) PER Protein & TIM Protein PERTIM Put them together stuck together HEY DUDE..they must interact with each other inside the cell!

After many experiments Discovered a clock-like loop PER & TIM genes are active until their proteins begin to bind to each other (heterodimer) They form complexes that enter the nucleus & shut down their own genes (with the help of Clock & Cycle) After a few hours - enzymes degrade the complexes & Viola..the cycle begins again!

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So…the SCN now known as “one of the main biological Clocks…however now we know that genes are involved also ….These genes have recently been identified in humans and rodents! Across the tree of life, from bacteria to humans, clocks use oscillating levels of proteins in feedback loops to keep time. Perhaps more amazing, fruit flies and mice — separated by nearly 700 million years of evolution — share the very same timekeeping proteins. — "First Runner Up: A Remarkable Year for Clocks," Science, 1998.

Biological Events Linked to Certain times in the 24 hour cycle 1:00 am – Pregnant women are most likely to go into labor 2:00 am – Levels of growth hormone are highest 4:00 am – Asthma attacks are most likely to occur 6:00 am – onset of menstruation is most likely to occur - Insulin levels lowest - BP & HR begin to rise 7:00 am Hayfever symptoms hi 8:00 am Risk for heart attack & stroke highest 6:00 pm urinary flow is highest 9:00 pm pain threshold lowest 11:00 allergic responses most likely