Lecture 2

Question 1

Where is the clock

Answer 1

Suprachiasmatic nuclei

Question 2

How many neurons does SCN contain

Answer 2

10,000

Question 3

Where are SCN found

Answer 3

Above optic chisma at base of 3rd ventricle

Question 4

How was SCN discovred at key

Answer 4

Lesioned the area, and it abolished rhythms in running, drinking and corticosterone

Question 5

Eye to SCN path

Answer 5

Retino-hypothalamic tract

Question 6

Experiements that proved clock

Answer 6

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

Question 7

Ralph experiment

Answer 7

Took WT SCN cellls and transplanted them into mutant short period. Mutant then adopted donors rhythm

Question 8

Herzog and Block experiment

Answer 8

Multi-electrode plate chip was cultured with SCN neurons and showed they have rhythmic activity

Question 9

Welsh and Reppert experiment

Answer 9

Single neruons were dispersed on a MEP and showed that they had rhythmic actiivty

Question 10

How are SCN cells coupled to each other

Answer 10

Gap junctions. When excitation was placed in one cell, spread to the other. Keeps the clock synchonised

Question 11

What gap junction proteins are expressed in the SCN

Answer 11

Connexins Cx32 and Cx36

Question 12

How was ‘clock’ gene found

Answer 12

Takahashi did a large mutant screen in mice and found clock gene, then cloned it 4 year later

Question 13

What mutation was shown in clock

Answer 13

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.

Question 14

Clock gene details

Answer 14

Expressed in SCN
Has a bHLH-PAS domain transcription factor
PAS domain in involved in protein protein interactions

Question 15

How was it know that clock needed a partner

Answer 15

bHLH domains normally act as dimers

Question 16

How was clock partner found

Answer 16

Using a yeast two hybrid protein interaction screen.

Question 17

What is the clock partner

Answer 17

BMAL1

Question 18

How was BMAL1 shown to be relevant

Answer 18

Knockout studies by Bradfields group

Question 19

How was per gene found

Answer 19

Know since 1984 about per in drosophhila, then it was clones 14 years later. 3 versions, mper 1,2,3

Question 20

mPER structure

Answer 20

Two PAS domains

Oscillate in the SCN

Question 21

Effects of mPER ko

Answer 21

mPER1 KO shows s short clock period
mPER 1 and 2 KO disrupt the clock
mPER 3 KO does not effect the clock

Question 22

How was cryptochrome 1 and 2 discovered

Answer 22

Homologous recombination of two mouse knockout genes. Pure luck. Double KO showed animals to be arrhythmic

Question 23

How was the tau hamster mutant protein found

Answer 23

Syntenic cloning and genetically directed representational difference analysis

Question 24

How does syntenic cloning work§

Answer 24

Take the WT and mutant, and do a subtractive hybridisation to find regions of different DNA. Then map these regions to a human

Question 25

Tau mutant gene in hamsters was

Answer 25

Casein kinase 1 epsilon. Mutation cause a decrease in the amplitude of mper1 RNA oscillations

Question 26

Summary of how the following were discovered

Clock
Tau
mper
cry1/2
bmal1

Answer 26

Clock - mutant screen
Tau - mutation analysis
mper cloned from drosphilla
cry1/2 by knockout study
bmal1 yeast two hybrid protein interaction screen

Question 27

Clock negative feedback loop

Answer 27

Clock gene
Cloc transcript - RNA
Clock protein, which then inhibits the clock gene

Question 28

Zeitgeber

Answer 28

External stimulus that sets a rhythm. In human clock, light is the zeitgeber and the changes in frequencies at dusk and dawn

Question 29

Circadian photoreception features

Answer 29

Sums light infomation over entire visual field
Insensitive to dim light
Insensitive to short durations of light
Sums light infomation over many minutes

Question 30

What is irradiance

Answer 30

Sums light over a entire visual field

Question 31

Can conless and rodless still entrain clock

Answer 31

Yes

Question 32

How was it worked out which cells are photosensitive to train clock

Answer 32

Injection of rabies virus which were photolabelled, and saw which cells tracted to the SCN

Question 33

What cells set the clock

Answer 33

Photosensitive retinal ganglion cells

Question 34

What wavelength do the cells respond best to

Answer 34

480nm. Blue light. Shows the biggest phase shift on action spectroscopy graph