Lecture 2 Flashcards
Where is the clock
Suprachiasmatic nuclei
How many neurons does SCN contain
10,000
Where are SCN found
Above optic chisma at base of 3rd ventricle
How was SCN discovred at key
Lesioned the area, and it abolished rhythms in running, drinking and corticosterone
Eye to SCN path
Retino-hypothalamic tract
Experiements that proved clock
Martin Ralph used a short period mutant hamster transplants
Herzog and Block cultured a SCN brain slice andshowed its rhythmic electrical activity
Welsh and Reppert showed that mist neruons in SCN contained a clock
Ralph experiment
Took WT SCN cellls and transplanted them into mutant short period. Mutant then adopted donors rhythm
Herzog and Block experiment
Multi-electrode plate chip was cultured with SCN neurons and showed they have rhythmic activity
Welsh and Reppert experiment
Single neruons were dispersed on a MEP and showed that they had rhythmic actiivty
How are SCN cells coupled to each other
Gap junctions. When excitation was placed in one cell, spread to the other. Keeps the clock synchonised
What gap junction proteins are expressed in the SCN
Connexins Cx32 and Cx36
How was ‘clock’ gene found
Takahashi did a large mutant screen in mice and found clock gene, then cloned it 4 year later
What mutation was shown in clock
A to T cause exon 19 to be spliced out. this removes large glutamine regions which are important for transcription. This makes it a weaker transcriptional activator.
Clock gene details
Expressed in SCN
Has a bHLH-PAS domain transcription factor
PAS domain in involved in protein protein interactions
How was it know that clock needed a partner
bHLH domains normally act as dimers
How was clock partner found
Using a yeast two hybrid protein interaction screen.
What is the clock partner
BMAL1
How was BMAL1 shown to be relevant
Knockout studies by Bradfields group
How was per gene found
Know since 1984 about per in drosophhila, then it was clones 14 years later. 3 versions, mper 1,2,3
mPER structure
Two PAS domains
Oscillate in the SCN
Effects of mPER ko
mPER1 KO shows s short clock period
mPER 1 and 2 KO disrupt the clock
mPER 3 KO does not effect the clock
How was cryptochrome 1 and 2 discovered
Homologous recombination of two mouse knockout genes. Pure luck. Double KO showed animals to be arrhythmic
How was the tau hamster mutant protein found
Syntenic cloning and genetically directed representational difference analysis
How does syntenic cloning work§
Take the WT and mutant, and do a subtractive hybridisation to find regions of different DNA. Then map these regions to a human
Tau mutant gene in hamsters was
Casein kinase 1 epsilon. Mutation cause a decrease in the amplitude of mper1 RNA oscillations
Summary of how the following were discovered
Clock Tau mper cry1/2 bmal1
Clock - mutant screen Tau - mutation analysis mper cloned from drosphilla cry1/2 by knockout study bmal1 yeast two hybrid protein interaction screen
Clock negative feedback loop
Clock gene
Cloc transcript - RNA
Clock protein, which then inhibits the clock gene
Zeitgeber
External stimulus that sets a rhythm. In human clock, light is the zeitgeber and the changes in frequencies at dusk and dawn
Circadian photoreception features
Sums light infomation over entire visual field
Insensitive to dim light
Insensitive to short durations of light
Sums light infomation over many minutes
What is irradiance
Sums light over a entire visual field
Can conless and rodless still entrain clock
Yes
How was it worked out which cells are photosensitive to train clock
Injection of rabies virus which were photolabelled, and saw which cells tracted to the SCN
What cells set the clock
Photosensitive retinal ganglion cells
What wavelength do the cells respond best to
480nm. Blue light. Shows the biggest phase shift on action spectroscopy graph
