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MCB 186CIRCADIAN BIOLOGY
The cellular-molecular mechanism of
the circadian clock CLOCK MUTANTS
Lecture #4 October 18, 2006J. W. Hastings
WHAT DO YOU DO WITH A MUTANT?
Isolate the gene responsible.
How do you map a gene? Clone the gene? How do you find out what the
gene does?
CIRCADIAN CLOCK MUTANTS
WHAT PHENOTYPES to SELECT?
The first CLOCK MUTANTS: the DROSOPHILA period (per) GENE
WILD TYPE~24 h
PER short~ 18 h
PER long~ 28 h
CIRCADIAN CLOCK GENES
1) DROSOPHILAper (PERIOD)tim (TIMELESS)
2) NEUROSPORAfrq (FREQUENCY)prd (PERIOD)
3) CYANOBACTERIAkai (CYCLE IN JAPANESE)
4) HAMSTERtau (PERIOD)
5) MOUSEclk (CLOCK)per1 (PERIOD)
6) ARABIDOPSIS (CCGs)toc1 (TIMING OF CAB)
lhy (LATE ELONG HYPOCOTYL)
cca1 (CIRC CLOCK ASSOCIATED)
The clock controls an overt rhythm in developmental potential in Neurospora.
movie courtesy of Van Gooch
FRQ (frequency) GENE IN NEUROSPORADunlap et al
CIRCADIAN CLOCK BIOCHEMICAL FEEDBACK CYCLE-NEUROSPORADUNLAP, 1999
LIGHT CAUSES PHASE SHIFTS BY INDUCTION OF FRQ mRNA
CROSTHWAITE, LOROS & DUNLAP, 1995
POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION
ClockProtein
ClockProtein
| |P P
ClockGene
ClockmRNA
PositiveRegulators
Other ClockProteins
| |P P
ATP
Figure 1b
CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS
The cyanobacterial clock
Kai genes
LUCIFERASE as a REPORTER for CIRCADIAN GENE EXPRESSION: INSERTED DOWNSTREAM from a
PROMOTER
Cyanobacterial in vivo Rhythm with Bacterial Luciferase as a Reporter
24016814412096720
Hours in Constant Light
2000
4000
6000
8000
12 60 84 108 132 156 180
228
192
204
10000
48
Lu
min
esce
nce
Kondo, Johnson Golden et al., 1993, PNAS
DARK PULSE PHASE SHIFTS IN CYANOBACTERIA KONDO ET AL 1993
BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATEDQ10 ~ 1.1
BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE
Dayphase
Nightphase
Codenumbers
LUCIFERASE REPORTER RANDOMLY INSERTED IN FRONT OF PROMOTERS IN
BACTERIA.
ABOUT 800 LUMINOUS COLONIES ISOLATED
ALL EXHIBITED CIRCADIAN RHYTHMS
ACROPHASES NOT ALL THE SAME
Liu et al., Circadian orchestration of gene expression in Cyanobacteria. (1995) Genes & Develop. 9: 1469-1478
CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,1995
CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,95
PERIOD MUTANTS OF CYANOBACTERIA KONDO ET AL.1995
CYANOBACTERIA ARHYTHYMIC & AMPLITUDE MUTANTS KONDO 1995
PERIOD VARIATION OF INDIVIDUAL CYANOBACTERIA MUTANT CLONES KONDO ET AL.1995
KAI genes in SYNECHOCOCCUS and MUTANT MAPPING Ishiura et al 1998 Science 281: 1519-1523
All of ~200 mutantsisolated map to thisgene cluster
PHENOTYPES OF KAI GENE INACTIVATIONS Ishiura et al 1998 Science 281: 1519-1523
EXPRESSION OF KAI GENE CONSTRUCTS Ishiura et al 1998 Science 281: 1519-1523
EXPRESSION and ROLES of KAI genes
Cyanobacterial Clockworks Model -1998
Circadian rhythms
C
CA
ABB
Protein Interactions
A B C
C
KaiC
B
KaiB
A
KaiA
rhythmicmRNAs
Kai proteins
kaiBCpkaiAp
Ishiura et al 1998 Science 281: 1519-1523
POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION
ClockProtein
ClockProtein
| |P P
ClockGene
ClockmRNA
PositiveRegulators
Other ClockProteins
| |P P
ATP
Figure 1b
EVIDENCE THAT TRANSCRIPTION and TRANSLATION are not
NECESSARY for CIRCADIAN OSCILLATIONS
IN CYANOBACTERIA Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedback
in circadian rhythm of KaiC phosphorylation Science 307: 251-254
NO RHYTHM OF kaiA or kaiBC mRNAs in DD
Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedbackin circadian rhythm of KaiC phosphorylation Science 307: 251-254
NO RHYTHM OF KaiC PROTEIN in DD Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254
RHYTHM OF KaiC PROTEIN PHOSPHORYLATION in DD Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254
Period Temperature Compensation of in vivo Rhythm of
KaiC Phosphorylation in DD
Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedbackin circadian rhythm of KaiC phosphorylation Science 307: 251-254
Period of phosphorylation rhythm corresponds to mutant period
Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254
Model for the posttranslational oscillator coupled with TTO
Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translationfeedbackin circadian rhythm of KaiC phosphorylation Science 307: 251-254
RHYTHM of Kai C PHOSPHORYLATION in a SOLUTION CONTAINING
only ATP plus Kai A, B and C
Nakajima, M. et al Kondo, T. (2005)Reconstitution of circadian oscillation of KaiC phosphorylation in vitro Science 308: 414-415
IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION
Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
In vitro phosphorylation rhythm is temperature compensated
Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
In vitro phosphorylation rhythm is temperature compensated
Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
Period of in vitro P-oscillation corresponds to mutant period
Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
Period of in vitro P-oscillation corresponds to mutant period
Nakajima, M et al Kondo,T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
HOW THE TWO COMPONENTS MAY RELATE
LBP mRNA DOES NOT CYCLE IN GONYAULAX
THE SINGLE CELLED ALGA ACETABULARIA MEDITERRANEA
2 ACETABULARIA RHYTHMS:O2 EVOL & CHLOROPLAST MOVEMENTSCHWEIGER ET AL, 1981
NUCLEUS IS IN ROOT- RHYTHM CONTINUES WHEN CUT OFFBUT A NEW NUCLEUS GRAFTED ON CONFERS ITS PHASE TO HOST
