W12L2 The circadian clock in Drosophila

Question 1

What is a circadian rhythms

Answer 1

-Biologicla rhythms of a day that are controlled by an internal oscillator

Question 2

Circadian rhythms in human

Answer 2

-at 9pm, melatonin secretion cause sleepiness
-stop of melatonin secretion in 07:30
-but there is genetic variation on the circadian clock

Question 3

Evidence of Self sustaining circadian clock

Answer 3

-release animal in a 24h of free run of light dark cycle or continuous dark
- have active and inactive period as if it is in a normal day. But there is shift in time due to circadian clock being faster

Question 4

How Circadian rhythms anticipate predictable daily environment

Answer 4

Young sunflowers reposition during the night to face the rising sun

Question 5

Circadian rhythms of gene expression

Answer 5

-circadian rhythm of gene expression with promoter: luciferase
-5-90% of gene are under circadian regulation. Half the gene on average

Question 6

Circadian clocks are entrained by the environment

Answer 6

-environmental input goes through an oscillator which lead to output
-Zeitgebers (time-keepers) such as light and temperature adjust the phase of the circadian oscillator

Question 7

Expirement on Light as a zeigeber (timekeeper)

Answer 7

• a normal organism is put into a all dark environment with pulse of light given
  -measure the oscillation
  -If light is given in the subjective day time, noting happen
  -if light is given at early night time, delay in ossicaltion. Late in the night, advance oscillation
  -entrainment allows organism to adapt to changing day length

Question 8

Circadian master clock

Answer 8

-In animals, ‘master’ clocks synchronise peripheral clocks
-photic zeitgeber influence master clock
-non photic zeitgeber influence peripheral and cellular osicillator
-synchronization occur

Question 9

What is a circadian oscillator?

Answer 9

Transcription-translation feedback loops (TTFL)

Question 10

How is the 24h cycle mainatin

Answer 10

-translational repression
-Codon bias
-Nuclear localization
-Phosphorylation
-Protein degration
-Transcriptional repression
-ncRNA

Question 11

Period, the first circadian mutant

Answer 11

• 3 alleles in the period mutant: per0 active all time, pers short period mutant, perl long droid mutant
  -3 mutant mapped to X chromosome
  -complementation test show that :per0 is null mutant, pers and perl is missense mutatant

Question 12

How does period gene works

Answer 12

-period forms a negative feedback loop
-a lag between the protein level and transcript abundant
-introducing the period gene (only coding region) can restore the oscillation can restore per0back to WT> period is self regulating

Question 13

timeless, a second circadian mutant

Answer 13

-rhythmic transcription
- tim arrhythmic mutant does not have the oscillation transcription

Question 14

Hyphothesis on tim and per

Answer 14

TIM and PER form a dimer to make a negative feedback loop to repress their own transcription
-TIM is needed for nuclear localization
-both tim and per does not have a DNA binding domain
-to additional arrhythmic mutants was discovered: clock and cycle, they are transcriptional activator of tim and per by binding to an E box

Question 15

Post transcriptional regulation in the circadian clock

Answer 15

1/ phosphorylation/ dephosphorylation
2/ protein degration
3/ translational control

Question 16

Phosphorylation regulation in the circadian clock

Answer 16

• phosphorylation / dephosphorylation: DBT (double time) is a kinase, phosphorilation of per allow it to interact with TIM
  -TIM and Per complex need to be phosphorolyse to enter the nucleases
  -SGG and CK2 phosphorylation TIM and PER (allow localization)
  -DBT phosphorylation CLK> release CLK CYC from e box
• Protein phosphatases (PP1 and PP2a) also act on TIM and PER
  -PP1/PP2a counteract SSG2/CK to inhibit nuclear localization of TIM-PER-DBT
  -acts as a speed bump to slow circadian clock

Question 17

Light-dependent degradation of TIM by Ubiquitin Proteasome

Answer 17

-Crytochrome is a blue light receptor, activation lead to interaction with TIM, cause TIM to disassociate with PER
-Crytochrome after use is degraded by JETLAG
-Jetlag is a F box which target Crytochrome for degradation by ubiquitination
-a phosphorylation event allow JET to interact with TIM
-Target of TIM for degradation, after that, DBT phosphorylation PER, causing it to be regconise by another F-box slimB. degredation of pER by UPS
-CLK-CYC can now bind to e box, activating transcription

Question 18

translational regulation of the circadian clock

Answer 18

-there is a 6-8 hours lag between per mRNA and protein
-due to degradation and control of translation, (transcription promoter TWF)
-mutant TWF have lower PER rhythms
-works via associating the 5’ cap with the PABP, stabilize the mRNA structure to promote translation