L35 Circadian Clocks

Question 1

Circa-diem

Answer 1

biological rhythms of about a day that are controlled by an internal occilator

Question 2

Circadian rhythm have a period of ~24hr in “free run”

Answer 2

Activity rhythms have a ~23hr period in continuous dark

Question 3

Circadian rhythm anticipates predictable daily environment

Answer 3

e.g. young sunflowers reposition during the night to face the rising sun

Question 4

Circadian rhythms of gene expression in all kingdoms of life

Answer 4

5-90% of genes are under circadian regulation

Question 5

Circadian clocks entrained by the environment

Answer 5

Input => oscillator => output e.g. gene expression, physiology, metabolism

Input e.g. light, temperature

Zeitgebers (time-keepers) such as light and temperature adjust phase of circadian oscillator

Question 6

Light is a zeitgeber that entrains circadian rhythm - phase response curve

Answer 6

see onenote

dead zone, phase delay, phase advance

Entrainment allows organisms to adapt to changing seasons (and recover from jetlag)

Question 7

Circadian rhythms are autonomous but…

Answer 7

see onenote

In animals, “master” clock synchronise peripheral clocks
- endogenous circadian rhythm

Question 8

Non-photic zeitgeber - 4 points

Answer 8

sleep-wake cycle
physical activity
social time
meals

Question 9

Circadian oscillators are -ve feedback loops

Answer 9

see onenote slides

Transcription-translation feedback loops (TTFL)

feedback loops results in rhythms of transcript abundance of the two genes e.g. GeneA and GeneB
- Gene A product activates gene B, and product of gene B represses gene A

layers of regulaiton

Question 10

Circadian rhythms in Drosophila

Answer 10

see onenote

Question 11

period - 3 mutants

Answer 11

see onenote slides

the first circadian mutant

Behavioural mutants - period mutant

• Altered eclosion rhythm
• Arrhythmic mutant
• Short period mutant
• Long period mutant

3 mutants mapped to X-chromosome

1. per0 = null = arrhythmic
2. perS = missense = short
3. perI = missense = long

Question 12

Eclosion

Answer 12

Eclosion = emergence of pupae

Question 13

period forms a feedback loop

Answer 13

see onenote slides

per mRNA cycles
PER protein cycles

Protein is not rhythmic but the mRNA was rhythmic
mRNA has self-regulation?

Question 14

timeless

Answer 14

second circadian mutant

Timeless mutants had similar phenotype to period mutants
- They were both involved somehow in the same pathway

Question 15

core negative feedback loop in Drosophila

Answer 15

TIM protein required for nuclear localisation of per protein

PER-TIM represses transcription of per gene

Period transcribed => period protein, reacts physically with timeless, allows period to enter the nucleus to repress its own expression

Timeless and period doesn’t have a DNA binding domain

Question 16

clock and cycle mutants

Answer 16

see onenote

clock (clk) and cycle (cyc) mutants are arrhythmic - transcriptional activators of per and tim genes

BUT, TTFL can be completed faster than 24hr so how is the 24 hour period maintained

Question 17

TTFL

Answer 17

Transcription-translation feedback loops (TTFL)

Question 18

Post-transcriptional regulation in the circadian clock

Answer 18

see onenote slides

1. phosphorylation/dephosphorylation
2. translational control
3. protein degradation

E-box element important for circadian regulation

DBT = double time

Question 19

Light-dependent degradation of TIM by ubiquitin proteasome

Answer 19

see onenote slide

Question 20

Light-dependent degradation of PER by ubiquitin proteasome

Answer 20

see onenote

Question 21

Translational regulation of circadian clock

Answer 21

see onenote slides

miRNA bantam targets clk 3’ UTR

Question 22

6-8 hour lag between per mRNA and PER protein - how?

Answer 22

see onenote

translational control

Question 23

Twenty-four gene

Answer 23

see onenote

TYF associates with 5’cap and PABP to promote translation

Twenty-four gene

• Twenty-four mutant = reduction of period protein levels but the mRNA is unaffected
• 24 seems to associate with polyA binding protein and Cap protein, suggests it is involved with the close-looped formation important for translation efficiency
• 24 promotes translations but its activity is delayed