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Mechanisms o f Development > Circadian Clocks > Flashcards

Circadian Clocks Flashcards

1
Q

why do all animals on earth mostly have circadian clocks?

A

because almost all animals in th world have been exposed to the chageing of light in the day and night and the changes that their body go through as a result o this changing in light

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2
Q

what is a circadian rhythm?

A

the metabolic cycles that occur within an organism over 24 hours

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3
Q

how can the changes in light alter reproduction and why would this happen?

A

reproductive systems of small animals drink during short periods of light and then turn n again in spring so do reproductive animals

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4
Q

what are the 5 basic features of a circadian clock?

A
  • the clock is an internal oscillator, enogenous tot he organism. This is how by the fact the clock continues to run in constant comditions.
  • the internal period os close to but not exactly 24 hours. (circadian means ‘about a day’)
  • the internal clock is set or entrained to the environment so that it runs with a 224 hour period
  • the internal clock is set or entrained to the environment so that it runs with a 24 hour period
  • light it the principal entraining signal
  • the clock is temperature compensated so that it doesn’t change relative to the weather
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5
Q

what demonstrates that the circadian clock is endogenous?

A

the oscillations in different metabolic process rates changes in relation to a circadian clock even in constant conditions

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6
Q

what type of systems in the human oscilate with circadian rhythms?

A
  • core body temp
  • melatonin
  • alertness
  • pituitary function
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7
Q

how can you measure a neutral clock within someone (that isn’t being entrained to the light to any other factors)

A

you can take someone and isolate them so that they have no sense of extneral time and measure their physiological conditions- you can measure when they eat, when they go to the toilet, when they wake and sleep.

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8
Q

what is the normal length of a young persons clock and have can this be viewed?

A
  • if you measure the circadian rhythm by measuring the time of wakefulness, sleep, meals , rectal temperature and evacuation of the bowls. and plot this in terms of time the day then you see that it moves to the right every day slightly- this suggests that the length of cycle is just over 24 hours- they wake later every day and so is around 24.5 hours
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9
Q

what does someone with a mutant long clock look like?

A
  • although their low body points occurs every 24.5 hours, the other systems run on a 48 hour clock- when you lock them up they think they have been in for half the time
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10
Q

what is interesting about the food intake of clock mutants?

A
  • even though they eat less overall because their clock runs on 48 hours, they do not lose weight. this suggests there may be link between metabolics and circadian cycles
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11
Q

what does a mutant short look like and what is interesting about this mutant ?

A

their clock runs less that 24 hours a day and want to get up earlier every day- they eat more frequently but dont put on weigh again a link between clock and metabolism

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12
Q

where did people originally think that the mammalian clock was and how did they test this? what did they find?

A

they originally thought that it was in rain so they took a rat brain and made systemic lesions to different parts of the brain and looked to see f the rat still had a circadian rhythm, did it still run on the wheel for the same time etc? they found that is ou burnt an area of the brain above the optic chasm then the rat became arrhythmic. this is an area of the brain called the suprchiasmatic nucleus

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13
Q

how was the suprachiasmatic nucleus thought to work as a clock for the body?

A
  • the idea was that there was a specific neural pathway from the ee to the base of the brain- dawn light stimulates the signal from your eye and stimulates the supracortical neuron- the stimulation of the upregulation of a clock gene called mper1, this then signals to and stimulates the
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14
Q

how is the stimulation of the retina and the supracortical nucleus thought to mediate the circadian rhythm?

A

light stimulates retina which stimulates the SCN to stimulate neural and humeral outputs in the day. when there is light the SCN also inhibits the pineal gland and prevents melatonin secretion which inhibits the SCN. But in the dark when the SCN isn’t activated to inhibit the pineal gland. melatonin is produced to inhibit the SCN and inhibit its neural and humour outputs.

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15
Q

hat is the evidence that the circadian rhythm is set via the eye?

A

people who have to have their eyes removed oftenare unable tot set their cock by light so need alarms etc and have physiological problems.

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16
Q

what animal is mostly sed for circadian rhythm analysis and why?

A

flies and drosophila are used because they can be easily mutagenised and their circadian rhythm can be checked via running wheel of activity.

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17
Q

what in the eye did people think was responsible for resetting the clock and how was this proved wrong?

A

people assumed that it was the rods and cones in the clock that were sending the signal but it isn’t. Even people who lose their rods and cones are still able to rest. it is instead set by ganglion cells at the front of the eye ch respond specifically to blue light in the morning and transmit a signal to the SCN

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18
Q

what cell in the eye detects light changes and signals to SCN?

A

the ganglion cells at the front of the eye

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19
Q

what is the name of the pigment in the ganglion cells that i stimulated by blue wavelength?

A

melanopsin

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20
Q

hat types of animals have very good clocks and why is this?

A

desert animals- if they come out too early they will get fried

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21
Q

what is a good way of finding genes involved in circadian rhythms?

A

do a mutagenic screen

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22
Q

why are flies and mice such massive models for screens?

A

they are good to do mutagenic screens in

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23
Q

what was the first screen which identified the first clock mutant?

A
  • a huge mouse screen was carried out with mice
  • they found a mutant that had a long cycle
  • they made a homozygous and found it was arhythmic!
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24
Q

how did they position the gene once they had found the homozygous mouse?

A

ye did a crossing and linking analysis by crossing with a SNP strain? and then they found a gene and did in situ and found it as expressed in the SCN- in the ‘right place’

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25
Q

what was the clock gene?

A

is a transcription factor found in the SCN

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26
Q

what region of the SCN showed it was a transcription activator? and which sown that it interacts with another protein ?

A
  • glutamine rich region

- PAS region

27
Q

how did the Gekeksi lab find the binding partner of clock protein?

A

they carried out a yeast- two-hybrid proteinn interaction screen- this involves expressing two plasmids: one has the protein of interest fused to the activation domain of transcriptional protein and the other plasmids is used to express many candidate genes which are also fused to the other domain of the TF. SO that when both are expressed in the same cell he yeast strain that is lacking of a required component but contains the component downstream of a UAS site, will only transcribe the required protein if the two proteins bind.

28
Q

what is a yeast- two hybrid protein interaction screen?

A

The most common screening approach is the yeast two-hybrid assay.[6] This system often utilizes a genetically engineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) is lacking. When grown on media that lacks these nutrients, the yeast fail to survive. This mutant yeast strain can be made to incorporate foreign DNA in the form of plasmids. In yeast two-hybrid screening, separate bait and prey plasmids are simultaneously introduced into the mutant yeast strain.

Plasmids are engineered to produce a protein product in which the DNA-binding domain (BD) fragment is fused onto a protein while another plasmid is engineered to produce a protein product in which the activation domain (AD) fragment is fused onto another protein. The protein fused to the BD may be referred to as the bait protein, and is typically a known protein the investigator is using to identify new binding partners. The protein fused to the AD may be referred to as the prey protein and can be either a single known protein or a library of known or unknown proteins. In this context, a library may consist of a collection of protein-encoding sequences that represent all the proteins expressed in a particular organism or tissue, or may be generated by synthesising random DNA sequences.[

29
Q

at protein was found to bind to clock?

A

BMAL1

30
Q

once the Gekakis lab had found that the binding partner of clock, how did the bradfield group sow s was essential for the clock to work?

A

they knocked tout and showed that you get an arhthmic mutant

31
Q

what was found to the be the consequence of breaking the block mechanism isn BMAL1 -/- mice?

A
  • they die younger
    they gain weight- sow shrinkage of internal organs
    show reduced lymphocyte numbers and altered immune function
  • the skin and the retina who major degeneration
32
Q

what technique did they use to look at the expression of clock within the eye and pineal gland?

A

they used an RNA detection assay bu running the gell with radioactive probe andt tis allow you to measure the level of RNA transcription - in the zebrafish

33
Q

what did they find when they ran an RNA assay on the clock levels in the eye and pineal gland?

A

they found that in both tissues there was a rhythm expression of clock in the zebrafish

34
Q

how did they discover that the clock mRnA was oscillating throughout the entire brain?

A

they wanted a control so they took a large area of the brain - forebrain, midbrain etc and found that they also oscillate

35
Q

after they found that the clock expression socialites throughout the entire brain, what did they find out about the rest of the body?

A

they found that clock oscillates throughout the entire fish body

36
Q

after they had found out about e expression of clock in the entire body, how did they show that this was really the case and not an artefact ?

A

they cultured the heart in neutral conditions and found thither was still an oscillation

37
Q

how did the show that the heart clock asiclations could in fact be entrained?

A

they cultured zebrafish heart organs and they put them on different light dark cycles and fond that they could be entrained

38
Q

after they found that the expression of clock in the heart could e entrained by light nt he heart, where else did they find this was possible?

A

in basically of the fish - the kidney, fins, spleen, liver, heart, gills

39
Q

how did the show in vivo that fish fans could be entrained by light and express different levels of clock?

A

they made a transgenic fish and expresses luciferase (short half life) downstream of a clock promoter and found that the luciferase glow oscilatted and could be entrained by light dark - in the light clock is expressed in the heart and when you put it in constant darkness the luminescence stops

40
Q

when over the 24 hours is clock expressed?

A

in the dark

41
Q

what is the current idea about the expression of clock in the body? in mammals?

A

peripheral tissues in mammals are thought to contain their own circadian pacemaker- the signal from the SCN is then needed to set and entrain the cycle

42
Q

how is the circadian clock thought to develop in the mama foetus?

A

materna circulating melatinin 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

43
Q

how was it tested when the circadian clock starts during zebrafish development?

A

they exposed eggs to a light dark cycle and looked at the expression of clock. They then compared this to the dark dar cycle embryos. They measured using an RNA expression assay. The first peak in the embryo of clock is found a few hours into the second day post fertilisation- there is a steady increase in the dar dark

44
Q

ow early were zebreafish found to respond to light dark cycles?

A

as early as 6 hours post fertilisation in the mid blastula stage!!

45
Q

what gene was shown to be light responsive even when there is no gastrulation?

A

crya1a

46
Q

how do you find out which genes in the fish are light responsive?

A

you do a transcriptome analysis of genes in the embryo in the light and in the dark and look at which ones change and this will show you which are light responsive

47
Q

what is the most activey turned on genes to lights?

A

DNA reapir enzymes- photolyases. The mother deposits these but then they run out and the fish uses light to turn on these repair genes.

48
Q

how early can zebrafish embryos transcribe photlyases in response to light?

A

6 hours post fertilisation

49
Q

why do you need photlyase repair mechanisms?

A

because UV induces cross linkage between thymidines which needs to be repaired as it can cause mutations the fish use the fact that there is light to trigger this repair response

50
Q

how was it shown that the fish need to be grown in light in order to survive.

A

sibling embryos and gave them a V light pulse and then put some on leith dark cycle and some just in the dark. if you raise embryos on a light dark cycle then they survive just as well as a non UV exposed embryo. but if you put them back into the dark then they dont survive as well- 80 die within first day because the dotnt have this DNA repair machinery

51
Q

how does fish lay egg time offer a selective advantage

A
  • if laid in the morning there is light which can activate these DNA lysases to repair DNA damage caused by UV
52
Q

order to prevent using entire zebrafish for these experiments, how can you get round this?

A

you can produce light sensitive zebrafish cell lines which you can use for experiments

53
Q

what is the characteristic of photosensitivity in tissues?

A

each tissue has different combinations of light photopigment molecules which it uses to give it phot sensitivity

54
Q

which process is incredibly regulated by light?

A

cell cycle progression- when the cells divide

55
Q

how can you measure cell division?

A

using BrdU which is incorporated into cells and those that have gone through S phase will light up blue when you apply label with an antibody

56
Q

if you use BrdU during development, what do you see? in fish, cell lines mammals

A

you find around 9 hours after fert you see that most cells replicate their DNA at around 6pm and then undergo mitosis at around 21 hours post fertilisation

57
Q

how can you measure mitosis?

A

you can label for molecules involved in mitosis or stain for phos histones- mitosis occurs just before dawn

58
Q

when does mitosis occur in fish and mammals?

A
  • repelicate DNA early evening and undergo mitosis just before dawn- you want as much cell cycle in the night to avoid UV damage and OD stress damage
59
Q

how can you look at the time of mitosis and a clock gene expression at the same time? what do

A

take cell cycle genes and a clock gene and link them to luciferase (different colour) and then you see that normally cyclin peaks just before the period peak and then you can jet lag the cells and see how his changes. You see the the clock gene seems to repress the ability of cells to enter G1

60
Q

what was the link between clock gene expression and regeneration and how was this tested?

A
  • cut the liver and then the liver regenrates- measure the stages of the cell cycle as the liver region - the division in this region liver was clock controlled and he figured out that there is a link in terms of regulation mitosis, a link between the clock and cycle cycle which is dependent not the clock driving the rhythm of one of the cell cycle regulators called Wee one which phosphorylates and regulates the activity of cyclin D1 - when this happens they dont enter mitosis and when it is switched off then they do
61
Q

what happens when get cancer?

do tumours know the time of day? in terms of light controlling time of cel division.

A
  • want to give drug when the cells are DNA replicating. because there is a lock in dveeloping tumours they respond differently at different times of day- when can maximally apply drugs to impact tumour- want to give while cells are replicating
    when optiammly timed cancer drug treatement get 22% cancer treatment
62
Q

do blind women get breast cancer?

A

no- maybe because of melatonin-

63
Q

how does shift work increase cancer risk?

A

woman that do if work has an 80% increase in great cancer-

64
Q

what are the three main ways of measuring gene expression levels?

A
  • microarray
  • qPCR
  • RNA seq

Mechanisms o f Development (15 decks)

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