Lecture 2 Flashcards

1
Q

Where is the clock

A

Suprachiasmatic nuclei

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2
Q

How many neurons does SCN contain

A

10,000

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3
Q

Where are SCN found

A

Above optic chisma at base of 3rd ventricle

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4
Q

How was SCN discovred at key

A

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

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5
Q

Eye to SCN path

A

Retino-hypothalamic tract

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6
Q

Experiements that proved clock

A

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

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7
Q

Ralph experiment

A

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

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8
Q

Herzog and Block experiment

A

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

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9
Q

Welsh and Reppert experiment

A

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

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10
Q

How are SCN cells coupled to each other

A

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

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11
Q

What gap junction proteins are expressed in the SCN

A

Connexins Cx32 and Cx36

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12
Q

How was ‘clock’ gene found

A

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

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13
Q

What mutation was shown in clock

A

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.

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14
Q

Clock gene details

A

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

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15
Q

How was it know that clock needed a partner

A

bHLH domains normally act as dimers

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16
Q

How was clock partner found

A

Using a yeast two hybrid protein interaction screen.

17
Q

What is the clock partner

18
Q

How was BMAL1 shown to be relevant

A

Knockout studies by Bradfields group

19
Q

How was per gene found

A

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

20
Q

mPER structure

A

Two PAS domains

Oscillate in the SCN

21
Q

Effects of mPER ko

A

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

22
Q

How was cryptochrome 1 and 2 discovered

A

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

23
Q

How was the tau hamster mutant protein found

A

Syntenic cloning and genetically directed representational difference analysis

24
Q

How does syntenic cloning work§

A

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

25
Tau mutant gene in hamsters was
Casein kinase 1 epsilon. Mutation cause a decrease in the amplitude of mper1 RNA oscillations
26
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 ```
27
Clock negative feedback loop
Clock gene Cloc transcript - RNA Clock protein, which then inhibits the clock gene
28
Zeitgeber
External stimulus that sets a rhythm. In human clock, light is the zeitgeber and the changes in frequencies at dusk and dawn
29
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
30
What is irradiance
Sums light over a entire visual field
31
Can conless and rodless still entrain clock
Yes
32
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
33
What cells set the clock
Photosensitive retinal ganglion cells
34
What wavelength do the cells respond best to
480nm. Blue light. Shows the biggest phase shift on action spectroscopy graph