HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM?
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MCB 186CIRCADIAN BIOLOGY
Slides Lecture 3 Clock genes & Biochemical Mechanisms
October 5, 2005
J. W. Hastings
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HOW DO YOU EXPERIMENTALLY PROBE FOR MECHANISM?
ALTER CONDITIONS (temperature, light cycle)ISOLATE MUTANTS (select for what?)
APPLY INHIBITORS OR DRUGS (what kinds?)
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TEMPERATURE HAS ONLY SMALL EFFECT ON PERIOD
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DROSOPHILA PER CLOCK MUTANTS
WILD TYPE~24 h
PER short~ 18 h
PER long~ 28 h
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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?
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CIRCADIAN CLOCK GENES
1) DROSOPHILAper (PERIOD)tim (TIMELESS)
2) NEUROSPORAfrq (FREQUENCY)prd (PERIOD)
3) CYANOBACTERIAkai (CYCLE IN JAPANESE)
4) ARABIDOPSIStoc1 (TIMING OF CAB)lhy (LATE ELONG HYPOCOTYL)cca1 (CIRC CLOCK ASSOCIATED)
5) MOUSEclk (CLOCK)per1 (PERIOD)
6) HAMSTERtau (PERIOD)
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The clock controls an overt rhythm in developmental potential in Neurospora.
movie courtesy of Van Gooch
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FRQ (frequency) GENE IN NEUROSPORADunlap et al
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Can we distinguish
CLOCK GENES from
CLOCK CONTROLLED GENES?
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CIRCADIAN CLOCK MUTANTS IN ARABIDOPSIS IDENTIFIED BY FIREFLY LUCIFERASE REPORTING
MILLER, CARRE. STRAYER, CHUA & KAY, 1995
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MODELING INPUT TO AND OUTPUT FROM THE CLOCK
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LUCIFERASE REPORTER for CIRCADIAN GENE EXPRESSION
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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 180228
192
204
10000
48
Lum
ines
cenc
e
Kondo, Johnson Golden et al., 1993, PNAS
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MEASURING ALL OR ONLY SOME CULTURES
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BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATEDQ10 ~ 1.1
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DARK PULSE PHASE SHIFTS IN CYANOBACTERIA KONDO ET AL 1993
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LIGHT CAUSES PHASE SHIFTS BY INDUCTION OF FRQ mRNA
CROSTHWAITE, LOROS & DUNLAP, 1995
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BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE
Dayphase
Nightphase
Codenumbers
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• Turntable Screening Apparatus: 12 positions for petri dishes or titer plates
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Screen Display–CCD Turntable Apparatus
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PERIOD MUTANTS OF CYANOBACTERIA KONDO ET AL.1995
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CYANOBACTERIA ARHYTHYMIC & AMPLITUDE MUTANTS KONDO 1995
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PERIOD VARIATION OF INDIVIDUAL CYANOBACTERIA MUTANT CLONES KONDO ET AL.1995
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MUTANTS V.S. WILD TYPE IN DIFFERENT LD CYCLES
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MUTANT TAU-28 V.S. WILD TYPE
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SURVIVAL CURVE FOR FLIES KEPT IN LIGHT/DARK CYCLES WITH PERIODS 20, 24 AND 28 HOURS
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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
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LUCIFERASE REPORTER for CIRCADIAN GENE EXPRESSION
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CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,1995
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CLASSES OF CLOCK-CONTROLLED GENES IN CYANOBACTERIA LIU,95
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KAI genes in SYNECHOCOCCUS and MUTANT MAPPING Ishiura et al 1998 Science 281: 1519-1523
All of ~200 mutantsisolated map to thisgene cluster
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PHENOTYPES OF KAI GENE INACTIVATIONS Ishiura et al 1998 Science 281: 1519-1523
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EXPRESSION OF KAI GENE CONSTRUCTS Ishiura et al 1998 Science 281: 1519-1523
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POSTULATED FEEDBACK LOOPS IN REGULATION OF CLOCK GENE EXPRESSION
ClockProtein
ClockProtein
| |P P
ClockGene
ClockmRNA
PositiveRegulators
Other ClockProteins
| |P P
ATP
Figure 1b
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COMMON ELEMENTS IN THE DESIGN OF CORE CIRCADIAN OSCILLATORS DUNLAP, 1999
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CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS
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Cyanobacterial Clockworks Model -1998
Circadian rhythms
CC
A
ABB
Protein Interactions
A B C
C
KaiC
BKaiB
A
KaiA
rhythmicmRNAs
Kai proteins
kaiBCpkaiAp
Ishiura et al 1998 Science 281: 1519-1523
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Cyanobacterial Clockwork Model
Xu et al., EMBO J. 22: 2117-2126, 2003
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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
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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
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NO RHYTHM OF KaiC PROTEIN in DD Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254
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RHYTHM OF KaiC PROTEIN PHOSPHORYLATION in DD Tomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254
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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
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Period of phosphorylation rhythm corresponds to mutant periodTomita, Nakajima, Kondo & Iwasaki (2005) Science 307: 251-254
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Model for the posttranslational oscillator coupled with TTO
Tomita, Nakajima, Kondo & Iwasaki (2005) No transcription-translation feedbackin circadian rhythm of KaiC phosphorylation Science 307: 251-254
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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 In vitro Science 308: 414-415
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IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION
Nakajima, M etal Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
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In vitro phosphorylation rhythm is temperature compensated
Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
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In vitro phosphorylation rhythm is temperature compensated
Nakajima, M et al Kondo, T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
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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
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Period of in vitro P-oscillation corresponds to mutant periodNakajima, M et al Kondo,T (2005) Reconstitution of circadian oscillation in vitro Science 308: 414-415
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HOW THE TWO COMPONENTS MAY RELATE
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THE ALGA ACETABULARIA MEDITERRANEA
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2 ACETABULARIA RHYTHMS:O2 EVOL & CHLOROPLAST MOVEMENTSCHWEIGER ET AL, 1981
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NUCLEUS IS IN ROOT- RHYTHM CONTINUES WHEN CUT OFFBUT A NEW NUCLEUS GRAFTED ON CONFERS ITS PHASE TO HOST