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  • Molecular Mechanisms of Circadian Clocks

  • Circadian Rhythms

    • Endogenous rhythm which persists under constant conditions

    • Period of approximately 24 hours • Entrainable (reset) by environmental cues

    such as light and temperature • Temperature compensated - period stays

    about the same at different temperatures

  • Circadian rhythm of locomoter activity

  • Body temperature cycle

  • Circadian rhythm of melatonin secretion

  • Circadian rhythms of photosynthetic genes in plants

  • Circadian rhythm of asexual reproduction in fungi Circadian rhythm of asexual reproduction in fungi

    24 hours of growth

    one circadian cyclePoint ofinoculation

    side

    top

    Neurospora

  • Suprachiasmatic Nucleus (SCN), the Master Pacemaker in Mammals

  • Science 2001 Feb 9;291(5506):1040-1043

    An hPer2 phosphorylation site mutation in familial advanced sleep phase

    syndrome (FASP). Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ, Fu YH. Department of Human Genetics, University of Utah

  • How does a clock work?

  • A B

    CD

    E OUTPUTINPUT gene expression enzyme activity hormone release

    Light Temperature

    OSCILLATOR

    Chemicals development behavior activity cycle sleep/wake cycle body temperature

  • frq frq frq frq frq frq temperature compensation,

    period length dominance/ recessivity affected

    linkageallele other clock properties

    temperature compensation, frq frq

    1

    2 3 4 6

    7

    8 9 arrhythmic/ recessive

    temperature compensation

    uncompensated entrainment nutritional compensation,

    cycloheximide resetting

    - isolated in the context of circadian rhythms

    period length dominance/ recessivity affected

    linkageallele other clock properties

    arg-13 cel

    Clock Genes

    mitochondrially related

    cyb-3 cyt-4 olir

    phe-1

    *

    *

    *

    temperature compensation,frq VII R arrhythmic/ recessive uncompensated

    entrainment nutritional compensation,*

    10

    Mutations Affecting the Neurospora Clock

    *

    *

    *

    * * *

    *

    *

    *

    Other mutations affecting clock period length

    21 - 19 recessiveI Rsemidominant16 hrsVII R 20 - 40 temperature compensationIV Rsemidominant19.3 hrsVII R

    cys-4 19IV Rsemidominant te24 hrsVII R mperature compensation cys-12 19I Rsemidominant19.3 hrsVII R

    semidominant19.3 hrsVII R glp-3(ff-1) II R 19 semidominant29 hrsVII R I L 19 recessive

    VII R 29 hrs semidominant VII R

    [mi-1] 18 - 19 cya -5 IV R 19 cyb-2 18

    II L 20 semidominantI R 20semidominant temperature compensationVI L 23.5 hrschr

    VII R 18 - 20recessive temperature compensationprd-1 III C 25.8 hrs recessiveprd-2 V R 25.5 hrs recessive temperature compensationprd-3 I C 25.1 hrs dominantprd-4 I R 18 hrs temperature compensation

    cla-1 I R/VII R 27 hrs semidominant temperature compensation

    * Extent of period effect is dependent on the growth mediumGenetically frq2 = frq4 = frq6 and frq7 = frq8 **

  • Common theme among circadian oscillators

    Negative Element(s) clock gene (s)

    Positive Element(s) (-)

    (+)

    Negative elements in circadian feedback loops: Synechococcus: kaiC Neurospora: FREQUENCY Drosophila: PERIOD and TIMELESS Mouse: mCRYs and mPERs

    Positive elements in circadian feedback loops Synechococcus: kaiA Neurospora: WHITE COLLAR-1 and WC-2 Drosophila: dCLOCK and CYCLE Mouse: CLOCK, NPAS2 and BMAL1

    PAS domain containing transcription factors

  • Definitions of a central component of a circadian clock

    • Mutations result in long, short period, or arrhythmic phenotypes

    • Deletion of the locus abolishes the rhythms • Both mRNA and protein cycle • Constitutive expression eliminates the clock • Feeds back negatively on its own expression • Change in levels resets the clock

  • Transcripts and Domains in frq and FRQ

    FRQ ORFs upstream ORFs

    989aa

    bp

    basic serine rich/acidic acidic TG/SG repeat

    coiled-coil1 k proline rich

    frq 3 frq 7,8 frq 1 frq 9 1 bp deletion

    frq 2,4,6 ala895->thrgly459->asp gly482->serglu364->lys

    (19.0 hr)(29.0 hr) (16.5 hr) (arrhythmic)(24.0 hr)

  • Both FRQ protein and frq RNA cycle

    f r q + m R N A

    2 0 3

    1 1 8

    F R Q

    1 1 1 8 2 5 4 7D D 3 3 4 01 5 2 2 2 9 3 6 4 3 5 1 1 81 5 h o u r s C T 0 8 1 6 1 6 4 84 1 2 2 0 0 4 1 2 2 08

    f r q + f r q 1 0

    0

    5

    1 0

    1 1 1 5 1 8 2 2 2 5 2 9 3 3 3 6 4 0 4 3 4 7 5 1 H o u r s i n C o n s t a n t D a r k n e s s

    5

    1 0

    0

  • The Neurospora (fungal) circadian feedback loops

    FRQ

    WC-1/WC-2

    frq

    +

    +

    WC-1

    WC-2

    -

    +

  • Circadian interlocked feedback loops in fruit fly and mouse

    mCrys mPers

    +

    + Bmal1 CLOCK/ NPAS2

    -BML/CLK/NPAS2 mCRYs mPERs

    Mammals

    dPer Tim

    +

    + dClock

    Cycle

    -CLK/CYC PER TIM

    Fruit fly

  • The positive feedback loops are important for the robustness of the clock

    FR Q

    Time Time

    low levels of WCs high levels of WCs

  • How do environmental signals reset clocks?

    Two most important signals: Light and Temperature

  • Circadian rhythm of locomoter activity

  • Light resetting of the Neurospora clock

    DD

    LD12/12

  • Light resets the Neurospora clock by induction of frq mRNA

    t i m e

    f r q R N A

    f r q R N A

    hours in dark - light

    frq

    0

    10

    20

    30

    40

    50

    60 fr

    q

    rib os

    om al

    R N

    A

    28 32 36 44 48 28 32 36 44 4840 40

    + light

    - light + light hours in the dark before a 2 minute light pulse

    28 32 36 40 44 48 28 32 36 40 44 48

  • Light resets the Drosophila clock by degrading TIMELESS protein

  • Light resets the mammalian clock by inducing Per RNA expression

    Dark

    Light

  • Cell

    Nucleus FRQ

    PP

    kinase

    turnover

    ?

    frq

    ccgs

    CCREfrq

    CCREccgs

    Clock-controlled processes

    FRQ

    Cell

    Nucleus

    CYC:CLK

    PER

    PP

    turnover ?

    per

    ccgs CCREccgs

    Clock-controlled processes

    TIM/PER tim

    E-box TIM

    E-box

    DBT

    Fungi LIGHT

    WC-2::WC-1

    -+ Insect

  • Circadian Rhythms Circadian rhythm of locomoter activity Body temperature cycle Circadian rhythm of melatonin secretion Circadian rhythms of photosynthetic genes in plants Suprachiasmatic Nucleus (SCN), the Master Pacemaker in Mammals An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome (FASP). How does a clock work? Common theme among circadian oscillators Definitions of a central component of a circadian clock Both FRQ protein and frq RNA cycle The Neurospora (fungal) circadian feedback loops Circadian interlocked feedback loops in fruit fly and mouse How do environmental signals reset clocks? Circadian rhythm of locomoter activity Light resetting of the Neurospora clock Light resets the Neurospora clock by induction of frq mRNA Light resets the Drosophila clock by degrading TIMELESS protein Light resets the mammalian clock by inducing Per RNA expression

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