molecular mechanisms of circadian clocksallele linkage other clock properties arg-13 cel clock genes...
TRANSCRIPT
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 asexual reproduction in fungiCircadian rhythm of asexual reproduction in fungi
24 hours ofgrowth
one circadian cyclePoint ofinoculation
side
top
Neurospora
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
AB
CD
E OUTPUTINPUT gene expressionenzyme activityhormone release
LightTemperature
OSCILLATOR
Chemicals developmentbehavioractivity cyclesleep/wake cyclebody temperature
frqfrqfrqfrqfrqfrq temperature compensation,
period length dominance/recessivity affected
linkageallele other clock properties
temperature compensation,frqfrq
1
2346
7
89 arrhythmic/ recessive
temperature compensation
uncompensatedentrainmentnutritional compensation,
cycloheximide resetting
- isolated in the context of circadian rhythms
period length dominance/recessivity affected
linkageallele other clock properties
arg-13cel
Clock Genes
mitochondriallyrelated
cyb-3cyt-4olir
phe-1
*
*
*
temperature compensation,frq VII R arrhythmic/ recessiveuncompensated
entrainmentnutritional compensation,*
10
Mutations Affecting the Neurospora Clock
*
*
*
***
*
*
*
Other mutations affecting clock period length
21 - 19 recessiveI Rsemidominant16 hrsVII R20 - 40 temperature compensationIV Rsemidominant19.3 hrsVII R
cys-4 19IV Rsemidominant te24 hrsVII R mperature compensationcys-12 19I Rsemidominant19.3 hrsVII R
semidominant19.3 hrsVII R glp-3(ff-1) II R 19semidominant29 hrsVII R I L 19 recessive
VII R 29 hrs semidominantVII R
[mi-1] 18 - 19cya -5 IV R 19cyb-2 18
II L 20semidominantI R 20semidominant temperature compensationVI L 23.5 hrschr
VII R 18 - 20recessive temperature compensationprd-1 III C 25.8 hrsrecessiveprd-2 V R 25.5 hrsrecessive temperature compensationprd-3 I C 25.1 hrsdominantprd-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)
PositiveElement(s) (-)
(+)
Negative elements in circadian feedback loops:Synechococcus: kaiCNeurospora: FREQUENCYDrosophila: PERIOD and TIMELESS Mouse: mCRYs and mPERs
Positive elements in circadian feedback loopsSynechococcus: kaiANeurospora: WHITE COLLAR-1 and WC-2Drosophila: dCLOCK and CYCLE Mouse: CLOCK, NPAS2 and BMAL1
PAS domain containingtranscription 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 ORFsupstream ORFs
989aa
bp
basic serine rich/acidic acidicTG/SG repeat
coiled-coil1 kproline rich
frq 3 frq 7,8 frq 1 frq 91 bp deletion
frq 2,4,6ala895->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 sC 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 1H 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
Circadian interlocked feedback loops in fruit fly and mouse
mCrysmPers
+
+Bmal1CLOCK/NPAS2
-BML/CLK/NPAS2
mCRYsmPERs
Mammals
dPerTim
+
+dClock
Cycle
-CLK/CYC
PERTIM
Fruit fly
The positive feedback loops are important for the robustness of the clock
FRQ
Time Time
low levels of WCs high levels of WCs
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
60fr
q
ribos
omal
RN
A
28 32 36 44 48 28 32 36 44 4840 40
+ light
- light + lighthours in the dark before a 2 minute light pulse
28 32 36 40 44 48 28 32 36 40 44 48
Cell
NucleusFRQ
PP
kinase
turnover
?
frq
ccgs
CCREfrq
CCREccgs
Clock-controlledprocesses
FRQ
Cell
Nucleus
CYC:CLK
PER
PP
turnover?
per
ccgsCCREccgs
Clock-controlledprocesses
TIM/PER tim
E-boxTIM
E-box
DBT
FungiLIGHT
WC-2::WC-1
-+ Insect