what do you do with a mutant?
DESCRIPTION
MCB 186 CIRCADIAN BIOLOGY The cellular-molecular mechanism of the circadian clock CLOCK MUTANTS Lecture #4 October 18, 2006 J. W. Hastings. CIRCADIAN CLOCK MUTANTS WHAT PHENOTYPES to SELECT?. WHAT DO YOU DO WITH A MUTANT?. Isolate the gene responsible. - PowerPoint PPT PresentationTRANSCRIPT
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MCB 186CIRCADIAN BIOLOGY
The cellular-molecular mechanism of
the circadian clock CLOCK MUTANTS
Lecture #4 October 18, 2006J. W. Hastings
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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?
CIRCADIAN CLOCK MUTANTS
WHAT PHENOTYPES to SELECT?
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The first CLOCK MUTANTS: the DROSOPHILA period (per) GENE
WILD TYPE~24 h
PER short~ 18 h
PER long~ 28 h
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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)
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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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CIRCADIAN CLOCK BIOCHEMICAL FEEDBACK CYCLE-NEUROSPORADUNLAP, 1999
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LIGHT CAUSES PHASE SHIFTS BY INDUCTION OF FRQ mRNA
CROSTHWAITE, LOROS & DUNLAP, 1995
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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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CORE CLOCK COMPONENTS IN FEEDBACK LOOPS OF 3 SYSTEMS
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The cyanobacterial clock
Kai genes
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LUCIFERASE as a REPORTER for CIRCADIAN GENE EXPRESSION: INSERTED DOWNSTREAM from a
PROMOTER
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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 84 108 132 156 180
228
192
204
10000
48
Lu
min
esce
nce
Kondo, Johnson Golden et al., 1993, PNAS
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DARK PULSE PHASE SHIFTS IN CYANOBACTERIA KONDO ET AL 1993
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BACTERIAL CIRCADIAN PERIOD is TEMP-COMPENSATEDQ10 ~ 1.1
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BACTERIAL COLONIES EXPRESSING BIOLUMINESCENCE
Dayphase
Nightphase
Codenumbers
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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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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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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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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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EXPRESSION and ROLES of KAI genes
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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
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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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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 period
Tomita, 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-translationfeedbackin 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 of KaiC phosphorylation in vitro Science 308: 414-415
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IN VITRO CIRCADIAN RHYTHM KaiC PHOSPHORYLATION
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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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 period
Nakajima, 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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LBP mRNA DOES NOT CYCLE IN GONYAULAX
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THE SINGLE CELLED 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