Circadian Clock Flashcards
What is the circadian clock?
It is an internal oscillator, “endogenous” to the organism
Give examples of factors that are variable and follow a circadian rhythm in humans?
- PRC = high day, low night
- Alertess = high day, low night
- Melatonin = low day, high night
- Cortisol = high day, low night
- Core body temp = high day, low night
What did Aschoff (1994) do?
Measured the sleep-wake cycle in 14 human subjects left in isolation from temporal cues. In 7 humans the the “free-running” circadian rhythms (e.g. the sleep-wake cycle and the rhythm of body temperature) remained internally synchronised (24.47hrs). In the remaining 7 subjects the sleep-wake cycle lengthened beyond 28 hr, desynchronizing from the rhythm of body temperature (internal desynchronization; mean sleep-wake cycle = 33.45 hr). It is concluded that a lengthening of the sleep-wake cycle (and of the wake time) results in a slowing down of the processes of digestion and evacuation of the bowels, in parallel with an apparent reduction of total energy expenditure.
- Subjects with extended sleep-wake cycle are clock mutants?
- A much rarer mutation is advanced sleep phase syndrom (ASPS). They have a naturally shorter clock.
Where is the master clock in mammals? And how was this discovered?
- Thought to be in the suprachiastmic nucleus (SCN). Adrenergic signals drive the release of melatonin from the pineal gland.
- This was found by during brain ablations in the rat.
What sets the SCN?
- The retina hypothalamic tract recieves the dawn/light signal and sets the SCN every morning.
- Blue light is recieved by retina cells and then communicated to the retina hypothalamic tract.
- The pathology of a blind person can determine their ability to maintain a circadian rhythm. If damage to the retinal hypothalamic tract is not the cause of blindness then they can still reset the SCN. Blind patients used to have their eyes removed but this led to “free-running.”
What did Takahshi (1993) find?
- From doing mutagenesis screens on mice the first clock mutant, called “clock” was discovered
- This clock mutant had a long period. When homozygous the mice became arythmic
- Four years later they positionally cloned the gene, clock - the first vertebrate clock gene
Describe the clock gene
- WISH found it to be expressed in the right place, SCN (one of the criteria of proof)
- It had the structure of a basic helix-looop-helix transcription factor. Large glutamine rich 3’ end.
- The mutation was a removal of exon 19 which removed a large part of Q-rich region.
- Glutamine regions are thought to be important for transcriptional activation of target genes
- Therefore, the clock mutation is a weaker transcriptional activator
- It also had a bHLH domain which means it bind to DNA and must have a partner
What did Gekakis (1998) find? What is a yeast two-hybrid protein interaction screen?
- They found the first Clock partner, BMAL1.
- Using a yeast two-hybrid protein interaction screen.
- This involves fusing a know protein to a transcription factor binding domain (the Clock protein) and then multiple other protein that wil be screened to the transcription factor active domain. When the two proteins interact then a reporter will be expressed.
- The transcription factor could be GAL4 which binds to UAS and the reporter GFP
- It is therefore a protein-fragment complementation assay
- These constructs are made in plasmids and transfected into yeast cells.
How did they prove the BMAL1 was an important clock protein? And what are the consequences of a BMAL1 mutant?
- They created a mouse knock-out. The animals become completely arythrimic
- Homozygous mouse die younger. Stop gaining weight. Internal organs shrink in size
- Cannot grow hair. Degeneration of the lens and cornea
How was it thought that lower vertebrates (zebrafish) control their circadian rhythm?
The eyes and the pineal gland
What was found about the clock gene in zebrafish? And then later flies and rats? What conclusions are now being drawn about mammals?
- RNA detection gel showed rhythmic expression of clock in the eye and the pineal gland BUT also throughout the entire brain and body!
- Light-entrainable clocks in zebrafish organs in culture and in vivo (luciferase assay).
- Transgenic animals of flies and rats also showed this to be the case in flies an rats
- Peripheral tissues in mammals are now thought to contain their own circadian pacemaker.
- A master clock SCN (in mammals) coordinates all the other clocks in the body. The mechanism underlying this is not well understood
How did the luciferase in vivo assay help?
- This method allowed for automated assays
- Fusion of luciferase gene (naturally glows) to the promoter of the gene of interest (clock)
What do we know about development of the circadian clock in mammals?
- Maternal circulating melatonin is thought to cross the placenta to set the embryonic SCN clock.
- In humans, however, this clock does not form functional output connections for about 9 weeks (post birth).
- Studying clock function is difficult in mammalian embryos as the embryo can not be easily accessed.
What do we know about circadian development in zebrafish?
- clock expression is first shown in 2dpf
- Respond to cry (other clock genes that respond to light - in the mouse, mPER and mCRY forma heterodimer that inhibits transcription mediated by another heterodimer formed by activators CLOCK and BMAL1) at early stages
- Zebrafish respond to light at early stages following mid-blastula transition. ~6hpf
- The DNA-repair enzyme, 6-4 photolyase, is maternally deposited and subsequently induced by light in early embryos
- This was indentified by transcriptome analysis
- Embryos have to see light early on to turn on DNA repair machinery. This is essential for survival.
- Was proved by testing survival of UV-treated embryos after being raised in LD or DD conditions.
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zebrafish embryos are much easier to study.
- transparent
- regularly lay eggs at 9:30 (when the lights come on)
- Develop ex utero
What is BrdU labelling?
- Cell progression can be monitored.
- BrdU is incorportated into the cell DNA in the S phase of cell replication
- BrdU can be passed to daughter cells upon replication.
- BrdU can then be stained for by antibody
- Will show any cells that have gone through S phase
